Atypical hemolytic uremic syndrome (AHUS) biomarker proteins

ABSTRACT

The disclosure provides biomarker proteins, a change in the concentration or activity level of which are associated with atypical hemolytic uremic syndrome (aHUS) or clinically meaningful treatment of aHUS with a complement inhibitor. Also provided are compositions and methods for interrogating the concentration and/or activity of one or more of the biomarker proteins in a biological fluid. The compositions and methods are useful for, among other things, evaluating risk for developing aHUS, diagnosing aHUS, determining whether a subject is experiencing the first acute presentation of aHUS, monitoring progression or abatement of aHUS, and/or monitoring response to treatment with a complement inhibitor or optimizing such treatment.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/453,268, filed Aug. 6, 2014, which claims priority to U.S.Provisional Application Nos. 61/913,180 and 61/863,299, filed Dec. 6,2013 and Aug. 7, 2013, respectively. The entire contents of theaforementioned application and any patents, patent applications, andreferences cited throughout this specification are herein incorporatedby reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 29, 2016, isnamed AXJ_176CN_Sequence.txt and is 12,514 bytes in size.

TECHNICAL FIELD

The field of the invention is medicine, immunology, molecular biology,and protein chemistry.

BACKGROUND

Hemolytic uremic syndrome (HUS) is characterized by thrombocytopenia,microangiopathic hemolytic anemia, and acute renal failure. HUS isclassified as one of two types: diarrheal-associated (D+ HUS; alsoreferred to as shiga toxin producing E. coli (STEC)-HUS or typical HUS)and non-diarrheal or atypical HUS (aHUS). D+ HUS is the most commonform, accounting for greater than 90% of cases and is caused by apreceding illness with a shiga-like toxin-producing bacterium, e.g., E.coli 0157:H7. aHUS is rare and has a mortality rate of up to 25%. Manypatients with this disease will sustain permanent neurological or renalimpairment, e.g., at least 50% of aHUS patients progress to end-stagerenal failure (ESRF). See, e.g., Kavanagh et al. (2006) British MedicalBulletin 77 and 78:5-22.

aHUS can be genetic, acquired, or idiopathic. Hereditable forms of aHUScan be associated with mutations in a number of human complementcomponents including, e.g., complement factor H (CFH), membrane cofactorprotein (MCP), complement factor I (CFI), C4b-binding protein (C4BP),complement factor B (CFB), and complement component 3 (C3). See, e.g.,Caprioli et al. (2006) Blood 108:1267-1279. Certain mutations in thegene encoding CD55, though not yet implicated in aHUS, are associatedwith the severity of aHUS. See, e.g., Esparza-Gordillo et al. (2005) HumMol Genet 14:703-712.

Until recently, treatment options for patients with aHUS were limitedand often involved plasma infusion or plasma exchange. In some cases,aHUS patients undergo uni- or bilateral nephrectomy or renaltransplantation (see Artz et al. (2003) Transplantation 76:821-826).However, recurrence of the disease in treated patients is common.Recently, treatment of aHUS patients with the drug Soliris® was approvedin the United States of America and in Europe. Despite finally having auseful drug for treatment of aHUS patients, there is still a need todiagnose patients with aHUS, as well as monitor the progression andabatement of aHUS.

SUMMARY

The present disclosure provides, among other things, a variety ofproteins whose activity and/or concentration in a biological fluid isabnormal in patients afflicted with aHUS and/or those aHUS patientsreceiving complement inhibitor therapy. Hereinafter these proteins arereferred to as “aHUS-associated biomarker proteins” or “aHUS biomarkerproteins”. For example, the inventors have observed that theconcentrations and/or activities of several proteins in the blood (e.g.,serum and/or plasma) and urine are abnormal in patients with aHUS. Theinventors have also observed that, following administration of anantagonist anti-C5 antibody (eculizumab) to a human, the concentrationsof a subset of these proteins change. In some instances, theconcentration of one or more of the proteins is normalized. While thedisclosure is not bound by any particular theory or mechanism of action,the inventors believe that monitoring a patient treated with acomplement inhibitor (such as an anti-C5 antibody) for a change inconcentration of one or more of these proteins—aHUS biomarkerproteins—is useful for, e.g., diagnosing a patient as having or at riskof developing aHUS. Monitoring the status of one or more of thesebiomarker proteins can also be useful for determining whether an aHUSpatient is responding to therapy with a complement inhibitor. Moreover,evaluating the status of one or more of the biomarkers is also usefulfor identifying a dose—a threshold dose—of a complement inhibitor, suchas an anti-C5 antibody, that by virtue of its effect on theconcentration of one or more of the aHUS biomarker proteins in the humanis sufficient to achieve a clinically-meaningful effect on the disease(i.e., sufficient to treat a complement-associated disease such asaHUS).

Accordingly, in one aspect, the disclosure features a method formonitoring or evaluating the status of atypical hemolytic uremicsyndrome (aHUS)-associated biomarker proteins in a subject (e.g., amammal such as a human) or a method for assessing one or both of theconcentration and activity level of at least one atypical hemolyticuremic syndrome (aHUS)-associated biomarker protein in a subject. Themethod comprises measuring in a biological fluid obtained from thesubject one or both of (i) the concentration of at least one (e.g., atleast two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20) aHUS-associated biomarker proteins in thebiological fluid, wherein the aHUS-associated biomarker proteins are anyof the biomarkers set forth in Table 1, e.g., one selected from thegroup consisting of: a proteolytic fragment of complement componentfactor B (e.g., Ba or Bb), soluble C5b9 (sC5b9), thrombomodulin, VCAM-1,von Willebrand Factor (vWF), soluble CD40 ligand (sCD40L), prothrombinfragment F1+2, D-dimer, CXCL10, MCP-1, TNFR1, IFN-γ, ICAM-1, IL-1 beta,IL-12 p70, complement component C5a, β2 microglobulin (β2M), clusterin,cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1),CXCL9, KIM-1, IL-18, vascular endothelial cell growth factor (VEGF),IL-6, albumin, IL-8, and CCL5. The subject can be, e.g., a human having,suspected of having, or at risk for developing, aHUS. The subject can beone who has been (or is being) treated with an inhibitor of complement(e.g., an inhibitor of complement component C5 such as an anti-C5antibody). The treatment can have occurred less than one month (e.g.,less than 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17,16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day) prior toobtaining the sample from the subject. The method can further includethe step of determining whether the subject has or is at risk ofdeveloping aHUS. Where the subject has been treated or is being treatedwith a complement inhibitor (e.g., an anti-C5 antibody) under apredetermined dosing schedule, the method can further includedetermining whether the patient is responsive (therapeutically) to thecomplement inhibitor therapy.

In another aspect, the disclosure features a method for monitoring orevaluating the status of atypical hemolytic uremic syndrome(aHUS)-associated biomarker proteins in a subject (e.g., a mammal suchas a human) or a method for assessing one or both of the concentrationand activity level of at least one atypical hemolytic uremic syndrome(aHUS)-associated biomarker protein in a subject. The method comprises:(A) measuring in a biological fluid obtained from the subject theconcentration of at least one (e.g., at least two, three, four, five,six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)aHUS-associated biomarker proteins in the biological fluid, wherein theaHUS-associated biomarker proteins are any of the biomarkers set forthin Table 1, e.g., one selected from the group consisting of: aproteolytic fragment of complement component factor B (e.g., Ba or Bb),soluble C5b9 (sC5b9), thrombomodulin, VCAM-1, von Willebrand Factor(vWF), soluble CD40 ligand (sCD40L), prothrombin fragment F1+2, D-dimer,CXCL10, MCP-1, TNFR1, IFN-γ, ICAM-1, IL-1 beta, IL-12 p70, complementcomponent C5a, P2 microglobulin (β2M), clusterin, cystatin C, NAG,TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), CXCL9, KIM-1,IL-18, vascular endothelial cell growth factor (VEGF), IL-6, albumin,IL-8, and CCL5; and (B) recording (e.g., in an electronic patientrecord) the results of the measurement(s) or communicating the resultsof the measurement(s) to the subject, the subject's guardian, or amedical professional in whose care the subject has been placed. Thesubject can be, e.g., a human having, suspected of having, or at riskfor developing, aHUS. The subject can be one who has been (or is being)treated with an inhibitor of complement (e.g., an inhibitor ofcomplement component C5 such as an anti-C5 antibody). The treatment canhave occurred less than one month (e.g., less than 31, 30, 29, 28, 27,26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9,8, 7, 6, 5, 4, 3, 2, or 1 day) prior to obtaining the sample from thesubject. The method can further include the step of determining whetherthe subject has or is at risk of developing aHUS. Where the subject hasbeen treated or is being treated with a complement inhibitor (e.g., ananti-C5 antibody) under a predetermined dosing schedule, the method canfurther include determining whether the patient is responsive(therapeutically) to the complement inhibitor therapy.

In yet another aspect, the disclosure features a method for monitoringor determining whether a patient is at risk for developing thromboticmicroangiopathy. The method includes (A) measuring in a biological fluidobtained from the subject the concentration of at least one (e.g., atleast two, three, four) biomarker protein associated with thrombosis orcoagulation in the biological fluid, wherein the biomarker proteins areany of such biomarkers set forth in Table 1 or Table 11, e.g., F1+2 orD-dimer; and (B) recording (e.g., in an electronic patient record) theresults of the measurement(s) or communicating the results of themeasurement(s) to the subject, the subject's guardian, or a medicalprofessional in whose care the subject has been placed. The subject canbe, e.g., a human having, suspected of having, or at risk fordeveloping, aHUS. The subject can be one who has been (or is being)treated with an inhibitor of complement (e.g., an inhibitor ofcomplement component C5 such as an anti-C5 antibody). The treatment canhave occurred less than one month (e.g., less than 31, 30, 29, 28, 27,26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9,8, 7, 6, 5, 4, 3, 2, or 1 day) prior to obtaining the sample from thesubject. The method can further include the step of determining whetherthe subject has or is at risk of developing aHUS (or confirming adiagnosis of aHUS) using any of the methods described herein. Where thesubject has been treated or is being treated with a complement inhibitor(e.g., an anti-C5 antibody) under a predetermined dosing schedule, themethod can further include determining whether the patient is responsive(therapeutically) to the complement inhibitor therapy, i.e., a reductionin the concentration of one or more of the thrombosis orcoagulation-associated biomarkers occurs following treatment with thecomplement inhibitor.

In another aspect, the disclosure features a method for monitoring orevaluating the status of atypical hemolytic uremic syndrome(aHUS)-associated biomarker proteins in a subject (e.g., a mammal suchas a human) or a method for assessing one or both of the concentrationand activity level of at least one atypical hemolytic uremic syndrome(aHUS)-associated biomarker protein in a subject. The method comprises:(A) measuring in a biological fluid obtained from the subject theconcentration of at least one (e.g., at least two, three, four, five,six, seven, eight, nine, 10, 11, 12, or 13) aHUS-associated biomarkerproteins in the biological fluid, wherein the aHUS-associated biomarkerproteins are any of the biomarkers set forth in Table 1, e.g., oneselected from the group consisting of: a proteolytic fragment ofcomplement component factor B (e.g., Ba or Bb), soluble C5b9 (sC5b9),C5a, thrombomodulin, VCAM-1, prothrombin fragment F1+2, D-dimer, sTNFR1,β2 microglobulin (β2M), clusterin, cystatin C, TIMP-1, and fatty acidbinding protein 1 (FABP-1); and (B) recording (e.g., in an electronicpatient record) the results of the measurement(s) or communicating theresults of the measurement(s) to the subject, the subject's guardian, ora medical professional in whose care the subject has been placed. Thesubject can be, e.g., a human having, suspected of having, or at riskfor developing, aHUS. The subject can be one who has been (or is being)treated with an inhibitor of complement (e.g., an inhibitor ofcomplement component C5 such as an anti-C5 antibody). The treatment canhave occurred less than one month (e.g., less than 31, 30, 29, 28, 27,26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9,8, 7, 6, 5, 4, 3, 2, or 1 day) prior to obtaining the sample from thesubject. The method can further include the step of determining whetherthe subject has or is at risk of developing aHUS. Where the subject hasbeen treated or is being treated with a complement inhibitor (e.g., ananti-C5 antibody) under a predetermined dosing schedule, the method canfurther include determining whether the patient is responsive(therapeutically) to the complement inhibitor therapy.

In some embodiments, any of the methods described herein can furthercomprise determining whether the subject has or is at risk fordeveloping aHUS. In some embodiments, an elevated concentration, ascompared to the concentration in a normal control biological fluid ofthe same type, of at least one of Ba, sC5b-9, C5a, sCD40L, prothrombinfragment F1+2, D-dimer, thrombomodulin, VCAM-1, vWF, FABP-1, β2M,clusterin, cystatin C, TIMP-1, albumin, NGAL, CXCL10, CXCL9, IL-18,TNFR1, VCAM-1, MCP-1, VEGF, CCL5, IL-6, IFNγ, indicates that the subjecthas, or is at risk for developing, aHUS.

In some embodiments, any of the methods described herein includedetermining whether the subject has responded to treatment with thecomplement inhibitor. In some embodiments, (a) a reduced concentration,as compared to the concentration in a sample of biological fluid of thesame type obtained from the subject prior to treatment with theinhibitor, of at least one of CXCL10, MCP-1, TNFR1, IFN-γ, a proteolyticfragment of complement component factor B (e.g., Ba or Bb), soluble C5b9(sC5b9), prothrombin fragment F1+2, d-dimer, thrombomodulin, VCAM-1, vonWillebrand Factor (vWF), complement component C5a, sC5b9, P2microglobulin (β2M), clusterin, cystatin C, NAG, TIMP-1, NGAL, fattyacid binding protein 1 (FABP-1), albumin, CXCL10, CXCL9, and KIM-1; or(b) an increased concentration, as compared to the concentration in asample of biological fluid of the same type obtained from the subjectprior to treatment with the inhibitor, of CCL5, indicates that thesubject is responsive to treatment with the inhibitor.

In another aspect, the disclosure features a method for monitoringresponsiveness of a subject (e.g., a mammal such as a human) totreatment with an inhibitor of complement component C5. The methodincludes: measuring the concentration of at least two aHUS-associatedbiomarker proteins in a biological fluid, wherein the aHUS-associatedbiomarker proteins are any of those set forth in Table 1, e.g., oneselected from the group consisting of: a proteolytic fragment ofcomplement component factor B (e.g., Ba or Bb), soluble C5b9 (sC5b9),thrombomodulin, VCAM-1, von Willebrand Factor (vWF), soluble CD40 ligand(sCD40L), prothrombin fragment F1+2, D-dimer, CXCL10, MCP-1, TNFR1,IFN-γ, ICAM-1, IL-1 beta, IL-12 p70, complement component C5a, β2microglobulin (β2M), clusterin, cystatin C, NAG, TIMP-1, NGAL, fattyacid binding protein 1 (FABP-1), CXCL9, KIM-1, IL-18, vascularendothelial cell growth factor (VEGF), IL-6, albumin, IL-8, and CCL5.The biological fluid is obtained from a subject: (i) having, suspectedof having, or at risk for developing, aHUS and (ii) who is being (or whohas been, e.g., recently) treated with an inhibitor of complementcomponent C5 under a predetermined dosing schedule. In accordance withsuch methods, (a) a reduced concentration, as compared to theconcentration in a sample of biological fluid of the same type obtainedfrom the subject prior to treatment with the inhibitor, of at least oneof CXCL10, MCP-1, TNFR1, IFN-γ, a proteolytic fragment of complementcomponent factor B (e.g., Ba or Bb), soluble C5b9 (sC5b9), prothrombinfragment F1+2, d-dimer, thrombomodulin, VCAM-1, von Willebrand Factor(vWF), complement component C5a, P2 microglobulin (β2M), clusterin,cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1),albumin, CXCL10, CXCL9, and KIM-1; or (b) an increased concentration, ascompared to the concentration in a sample of biological fluid of thesame type obtained from the subject prior to treatment with theinhibitor, of CCL5, indicates that the subject is responsive totreatment with the inhibitor.

In some embodiments, any of the methods described herein includedetermining whether the subject has responded to treatment with thecomplement inhibitor. In some embodiments, a reduced concentration, ascompared to the concentration in a sample of biological fluid of thesame type obtained from the subject prior to treatment with theinhibitor, of at least one of a proteolytic fragment of complementcomponent factor B (e.g., Ba or Bb), soluble C5b9 (sC5b9), C5a,thrombomodulin, VCAM-1, prothrombin fragment F1+2, D-dimer, sTNFR1, (32microglobulin (β2M), clusterin, cystatin C, TIMP-1, and fatty acidbinding protein 1 (FABP-1).

In another aspect, the disclosure features a method for monitoringresponsiveness of a subject to treatment with an inhibitor ofcomplement, wherein the method comprises: determining the concentrationof at least two aHUS-associated biomarker proteins in a biological fluidobtained from the subject, wherein the aHUS-associated biomarkerproteins are selected from the group consisting of: CXCL10, MCP-1,TNFR1, IFN-γ, IL-6, a proteolytic fragment of complement componentfactor B (e.g., Ba or Bb), soluble C5b9 (sC5b9), prothrombin fragmentF1+2, d-dimer, thrombomodulin, VCAM-1, von Willebrand Factor (vWF),complement component C5a, P2 microglobulin (β2M), clusterin, cystatin C,NAG, TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), albumin,CXCL9, KIM-1, and CCL5. The subject has, is suspected of having, or isat risk for developing aHUS and the subject has been or is being treatedwith an inhibitor of complement. (A) a reduced concentration, ascompared to the concentration in a sample of biological fluid of thesame type obtained from the subject prior to treatment with theinhibitor, of at least one of CXCL10, MCP-1, TNFR1, IFN-γ, IL-6, aproteolytic fragment of complement component factor B (e.g., Ba or Bb),soluble C5b9 (sC5b9), prothrombin fragment F1+2, d-dimer,thrombomodulin, VCAM-1, von Willebrand Factor (vWF), complementcomponent C5a, P2 microglobulin (β2M), clusterin, cystatin C, NAG,TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), albumin, CXCL9, andKIM-1; or (B) an increased concentration, as compared to theconcentration in a sample of biological fluid of the same type obtainedfrom the subject prior to treatment with the inhibitor, of CCL5,indicates that the subject is responsive to treatment with theinhibitor.

In yet another aspect, the disclosure features a method for reducing thenumber, frequency, or occurrence, likelihood of occurrence, or risk ofdeveloping, TMA, using a complement inhibitor in a manner sufficient toinduce a physiological change in at least two biomarker proteinsassociated with thrombosis or coagulation. The method includes: (a)determining the concentration of at least two biomarker proteins in abiological fluid obtained from the subject, wherein the biomarkerproteins are selected from Table 1 or 11 and relate to thrombosis and/orcoagulation (e.g., D-dimer or F1+2); and (b) administering to a subjecthaving, suspected of having, or at risk for developing, TMA an inhibitorof complement in an amount and with a frequency sufficient to cause aphysiological change in at least each of two (2) of the biomarkerproteins, wherein the physiological change is a reduction in theconcentration of the at least two biomarker proteins relative to theconcentration of the markers in an equivalent biological sample obtainedfrom the subject prior to treatment with the complement inhibitor. Themethod can include both measuring the concentration of the biomarkersbefore and after treatment.

In yet another aspect, the disclosure features a method for determiningwhether an aHUS patient treated with a complement inhibitor under apredetermined dosing schedule is in need of: (i) treatment with adifferent complement inhibitor or (ii) treatment with the samecomplement inhibitor under a different dosing schedule. The methodcomprises: (A) determining whether the aHUS patient is responsive totreatment with the complement inhibitor under the predetermined dosingschedule, wherein the determining comprises: measuring in a biologicalfluid obtained from the subject one or both of the concentration andactivity of at least two aHUS-associated biomarker proteins in thebiological fluid, wherein the aHUS-associated biomarker proteins areselected from the group consisting of: a proteolytic fragment ofcomplement component factor B (e.g., Ba or Bb), soluble C5b9 (sC5b9),thrombomodulin, VCAM-1, von Willebrand Factor (vWF), soluble CD40 ligand(sCD40L), prothrombin fragment F1+2, D-dimer, CXCL10, MCP-1, TNFR1,IFN-γ, ICAM-1, IL-1 beta, IL-12 p70, complement component C5a, P2microglobulin (β2M), clusterin, cystatin C, NAG, TIMP-1, NGAL, fattyacid binding protein 1 (FABP-1), CXCL9, KIM-1, IL-18, vascularendothelial cell growth factor (VEGF), IL-6, albumin, IL-8, and CCL5,and wherein: (a) a reduced concentration, as compared to theconcentration in a sample of biological fluid of the same type obtainedfrom the subject prior to treatment with the inhibitor, of at least oneof CXCL10, MCP-1, TNFR1, IFN-γ, a proteolytic fragment of complementcomponent factor B (e.g., Ba or Bb), soluble C5b9 (sC5b9), prothrombinfragment F1+2, d-dimer, thrombomodulin, VCAM-1, von Willebrand Factor(vWF), complement component C5a, sC5b9, P2 microglobulin (β2M),clusterin, cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1(FABP-1), albumin, CXCL10, CXCL9, and KIM-1; or (b) an increasedconcentration, as compared to the concentration in a sample ofbiological fluid of the same type obtained from the subject prior totreatment with the inhibitor, of CCL5, indicates that the subject isresponsive to treatment with the inhibitor; and (B) if the patient isnot responsive to treatment with the complement inhibitor, administeringthe patient a different complement inhibitor or the same complementinhibitor at a higher dose or more frequent dosing schedule as comparedto the predetermined dosing schedule.

In yet another aspect, the disclosure features a method for determiningwhether an aHUS patient treated with a complement inhibitor under apredetermined dosing schedule is in need of: (i) treatment with adifferent complement inhibitor or (ii) treatment with the samecomplement inhibitor under a different dosing schedule. The methodcomprises: (A) determining whether the aHUS patient is responsive totreatment with the complement inhibitor under the predetermined dosingschedule, wherein the determining comprises: measuring in a biologicalfluid obtained from the subject one or both of the concentration andactivity of at least two aHUS-associated biomarker proteins in thebiological fluid, wherein the aHUS-associated biomarker proteins areselected from the group consisting of: a proteolytic fragment ofcomplement component factor B (e.g., Ba or Bb), soluble C5b9 (sC5b9),C5a, thrombomodulin, VCAM-1, prothrombin fragment F1+2, D-dimer, sTNFR1,β2 microglobulin (β2M), clusterin, cystatin C,TIMP-1, and fatty acidbinding protein 1 (FABP-1), and wherein: (a) a reduced concentration, ascompared to the concentration in a sample of biological fluid of thesame type obtained from the subject prior to treatment with theinhibitor, of at least one of a proteolytic fragment of complementcomponent factor B (e.g., Ba or Bb), soluble C5b9 (sC5b9), C5a,thrombomodulin, VCAM-1, prothrombin fragment F1+2, D-dimer, sTNFR1, β2microglobulin (β2M), clusterin, cystatin C, TIMP-1, and fatty acidbinding protein 1 (FABP-1) indicates that the subject is responsive totreatment with the inhibitor; and (B) if the patient is not responsiveto treatment with the complement inhibitor, administering the patient adifferent complement inhibitor or the same complement inhibitor at ahigher dose or more frequent dosing schedule as compared to thepredetermined dosing schedule.

The concentration of one or more of the proteins can be measured using,e.g., an immunoassay (e.g., enzyme linked immunosorbent assay (ELISA), aradioimmunoassay (RIA), Western blotting, or dot blotting) or cytometricbead array (CBA; see the working examples). Such methods as well as kitsuseful for performing the methods are described herein. Suitable methodsfor measuring the activity of vWF are known in the art and describedherein.

In some embodiments of any of the methods described herein, theconcentrations of at least five individual aHUS-associated biomarkerproteins are measured. In some embodiments of any of the methodsdescribed herein, the concentrations of at least ten individualaHUS-associated biomarker proteins are measured. In some embodiments ofany of the methods described herein, the concentrations of at least 15individual aHUS-associated biomarker proteins are measured. In someembodiments of any of the methods described herein, the concentrationsof at least 20 individual aHUS-associated biomarker proteins aremeasured.

In some embodiments of any of the methods described herein, thebiological fluid is blood. In some embodiments, the biological fluid isa blood fraction, e.g., serum or plasma. In some embodiments, thebiological fluid is urine. In some embodiments of any of the methodsdescribed herein, all of the measurements are performed on onebiological fluid. In some embodiments of any of the methods describedherein, measurements are performed on at least two different biologicalfluids obtained from the subject. In some embodiments, theconcentrations of at least two individual aHUS-associated biomarkerproteins are measured and the concentration of the first aHUS-associatedbiomarker protein is measured in one type of biological fluid and thesecond aHUS-associated biomarker protein is measured in a second type ofbiological fluid.

In some embodiments of any of the methods described herein, theconcentrations of at least two (e.g., at least three, four, or all) ofIFN-γ, ICAM-1, IL-1 beta, and IL-12 p70 are measured. In someembodiments of any of the methods described herein, the concentrationsof both Ba and sC5b9 are measured. In some embodiments of any of themethods described herein, the concentrations of one or both of C5a andC5b9 are measured. In some embodiments of any of the methods describedherein, the concentrations of at least two (e.g., at least three, four,five, six, or all) of β2M, clusterin, cystatin C, NAG, TIMP-1, NGAL, andFABP-1 are measured. In some embodiments of any of the methods describedherein, the concentrations of CXCL10, CXCL9, and/or KIM-1 are measured.In some embodiments of any of the methods described herein, theconcentrations of one or both of D-dimer and F1+2 are measured. In someembodiments of any of the methods described herein, the concentrationsof at least two (e.g., at least three, four, or all) of sCD40L,prothrombin fragment F1+2, and D-dimer, are measured. In someembodiments of any of the methods described herein, the concentrationsof thrombomodulin, VCAM-1, and/or vWF are measured. In some embodimentsof any of the methods described herein, the concentrations of CXCL10,MCP-1, and/or TNFR1 are measured. In some embodiments of any of themethods described herein, the concentrations of at least two (e.g., atleast three, four, or all) of IFN-γ, ICAM-1, IL-1 beta, and IL-12 p70are measured.

In some embodiments of any of the methods described herein, theconcentrations of one or more of CXCL9, CXCL10, IL-1 beta, IL-12 p′70,IFN-γ, MCP-1, CCL5, sCD40L, and/or sTNFR1 is measured in the serum ofthe subject. In some embodiments, the concentrations of one or more ofcomplement component C5a, sC5b9, P2 microglobulin (β2M), clusterin,cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1),CXCL10, CXCL9, and/or KIM-1 are measured in the urine of the subject. Insome embodiments of any of the methods described herein, theconcentrations of one or more of NGAL, a proteolytic fragment ofcomplement component factor B (e.g., Ba or Bb), soluble C5b9 (sC5b9),prothrombin fragment F1+2, D-dimer, thrombomodulin, and/or vonWillebrand Factor (vWF) are measured in the plasma of the subject.

In some embodiments, the concentrations of two or more (e.g., three,four, five, six, seven, eight, nine, 10, 11, 12, or 13) of a proteolyticfragment of complement component factor B (e.g., Ba or Bb), soluble C5b9(sC5b9), C5a, thrombomodulin, VCAM-1, prothrombin fragment F1+2,D-dimer, sTNFR1, P2 microglobulin (β2M), clusterin, cystatin C, TIMP-1,and fatty acid binding protein 1 (FABP-1) are measured.

In some embodiments of any of the methods described herein, theconcentration of at least two of the group consisting of Ba, sC5b-9, andC5a is measured. In some embodiments of any of the methods describedherein, the concentration of one or both of Ba and sC5b9 is measured. Insome embodiments of any of the methods described herein, theconcentration of one or both of C5a and C5b9 are measured. In someembodiments of any of the methods described herein, the concentrationsof at least two individual members of the group consisting of β2M,clusterin, cystatin C, albumin, TIMP-1, NGAL, and FABP-1 are measured.In some embodiments of any of the methods described herein, theconcentrations of at least two individual members of the groupconsisting of CXCL10, CXCL9, IL-18, MCP-1, TNFR1, VEGF, IL-6, and IFNγare measured. In some embodiments of any of the methods describedherein, the concentration of one or both of d-dimer and F1+2 ismeasured. In some embodiments of any of the methods described herein,the concentrations of at least two individual members of the groupconsisting of sCD40L, prothrombin fragment F1+2, and d-dimer aremeasured. In some embodiments of any of the methods described herein,the concentration of thrombomodulin, VCAM-1, or vWF is measured. In someembodiments of any of the methods described herein, the concentration ofTNFR1 is measured. In some embodiments of any of the methods describedherein, the concentrations of at least two individual members of thegroup consisting of IFN-γ, CXCL10, CXCL9, IL-18, TNFR1, VCAM-1, MCP-1,VEGF, CCL5, and IL-6 are measured. In some embodiments of any of themethods described herein, the concentration of at least oneaHUS-associated biomarker protein selected from the group consisting ofIFN-γ, CXCL10, CXCL9, IL-18, TNFR1, VCAM-1, MCP-1, VEGF, and IL-6 ismeasured. In some embodiments of any of the methods described herein,the concentration of at least one aHUS-associated biomarker selectedfrom the group consisting of β2 microglobulin (β2M), clusterin, cystatinC, NAG, TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), CXCL10,CXCL9, albumin, and KIM-1 is measured. In some embodiments of any of themethods described herein, the concentration of at least oneaHUS-associated biomarker protein selected from the group consisting of:CXCL10, CXCL9, IL-18, MCP-1, TNFR1, VEGF, IL-6, CCL5, IFNγ, IL-8,ICAM-1, IL-1 beta, and IL-12 p70 is measured. In some embodiments of anyof the methods described herein, the concentration of CXCL9, CXCL10,IL-1 beta, IL-12 p′70, IFN-γ, MCP-1, CCL5, sCD40L, or sTNFR1 is measuredin the serum of the subject. In some embodiments of any of the methodsdescribed herein, the concentration of at least one aHUS-associatedbiomarker selected from the group consisting of β2 microglobulin (β2M),clusterin, cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1(FABP-1), CXCL10, CXCL9, albumin, and KIM-1 is measured in the urine ofthe subject. In some embodiments of any of the methods described herein,the concentration of NGAL, a proteolytic fragment of complementcomponent factor B (e.g., Ba or Bb), soluble C5b9 (sC5b9), prothrombinfragment F1+2, D-dimer, thrombomodulin, or von Willebrand Factor (vWF)is measured in the plasma of the subject. In some embodiments of any ofthe methods described herein, the concentration of Ba is measured (e.g.,in the plasma sample obtained from the subject).

In some embodiments of any of the methods described herein, the methodrequires recording the measured value(s) of the concentration of the atleast one aHUS biomarker protein. The recordation can be written or on acomputer readable medium. The method can also include communicating themeasured value(s) of the concentration of the at least one aHUSbiomarker protein to the subject and/or to a medical practitioner inwhose care the subject is placed.

In some embodiments, any of the methods described herein can include thestep of administering to the subject the complement inhibitor at ahigher dose or with an increased frequency of dosing, relative to thepredetermined dosing schedule, if the subject is not responsive totreatment with the inhibitor under the predetermined dosing schedule.

In some embodiments of any of the methods described herein, thecomplement inhibitor is administered to the subject under apredetermined dosing schedule based, in part, on the body weight of thesubject. For example, in the case of an antagonist anti-C5 antibody(e.g., eculizumab), for subjects having a body weight greater than orequal to 40 kg, the antibody can be administered to the subject for atleast 7 weeks under the following schedule: at least 800 mg of theantibody, once per week for four consecutive weeks; at least 800 mg ofthe antibody once during the fifth week; and at least 800 mg of theantibody bi-weekly thereafter. In some embodiments, the antibody isadministered to the subject for at least 7 weeks under the followingschedule: at least 900 mg of the antibody, once per week for fourconsecutive weeks; at least 1200 mg of the antibody once during thefifth week; and at least 1200 mg of the antibody bi-weekly thereafter.

In some embodiments of any of the methods described herein, for subjectshaving a body weight less than 40 kg but greater than or equal to 30 kg,the antibody can administered to the subject for at least 7 weeks underthe following schedule: at least 500 mg of the antibody, once per weekfor two consecutive weeks; at least 700 mg of the antibody once duringthe third week; and at least 700 mg of the antibody bi-weeklythereafter. In some embodiments, the antibody is administered to thesubject for at least 5 weeks under the following schedule: at least 600mg of the antibody, once per week for two consecutive weeks; at least900 mg of the antibody once during the third week; and at least 900 mgof the antibody bi-weekly thereafter.

In some embodiments of any of the methods described herein, the bodyweight of the subject is less than 30 kg, but is greater than or equalto 20 kg and the antibody is administered to the subject for at least 5weeks under the following schedule: at least 500 mg of the antibody,once per week for two consecutive weeks; at least 500 mg of the antibodyonce during the third week; and at least 500 mg of the antibodybi-weekly thereafter. In some embodiments, the antibody is administeredto the subject for at least 5 weeks under the following schedule: atleast 600 mg of the antibody, once per week for two consecutive weeks;at least 600 mg of the antibody once during the third week; and at least600 mg of the antibody bi-weekly thereafter.

In some embodiments of any of the methods described herein, the bodyweight of the subject is less than 20 kg, but is greater than or equalto 10 kg and the antibody is administered to the subject for at least 4weeks under the following schedule: at least 500 mg of the antibody oncea week for one week; at least 200 mg of the antibody once during thesecond week; and at least 200 mg of the antibody bi-weekly thereafter.In some embodiments, the antibody is administered to the subject for atleast 4 weeks under the following schedule: at least 600 mg of theantibody once a week for one week; at least 300 mg of the antibody onceduring the second week; and at least 300 mg of the antibody bi-weeklythereafter.

In some embodiments of any of the methods described herein, the bodyweight of the subject is less than 10 kg, but is greater than or equalto 5 kg and the antibody is administered to the subject for at least 5weeks under the following schedule: at least 200 mg of the antibody,once per week for one week; at least 200 mg of the antibody once duringthe second week; and at least 200 mg of the antibody once every threeweeks thereafter. In some embodiments, the antibody is administered tothe subject for at least 5 weeks under the following schedule: at least300 mg of the antibody, once per week for one week; at least 300 mg ofthe antibody once during the second week; and at least 300 mg of theantibody every three weeks thereafter. Additional exemplary anti-C5antibody dosing schedules (e.g., chronic dosing schedules) for aHUS aredescribed in International patent application publication no. WO2010/054403 (e.g., Tables 1 and 2 of WO 2010/054403), the disclosure ofwhich is incorporated herein by reference in its entirety.

In some embodiments of any of the methods described herein, theinhibitor is antibody or an antigen binding fragment thereof, a smallmolecule, a polypeptide, a polypeptide analog, a peptidomimetic, or anaptamer. In some embodiments, the inhibitor can be one that inhibits oneor more of complement components C1, C2, C3, C4, C5, C6, C7, C8, C9,Factor D, Factor B, properdin, MBL, MASP-1, MASP-2, or biologicallyactive fragments of any of the foregoing. In some embodiments of any ofthe methods described herein, the complement inhibitor inhibits one orboth of the generation of the anaphylatoxic activity associated with C5aand/or the assembly of the membrane attack complex associated with CSb.

The compositions can also contain naturally occurring or soluble formsof complement inhibitory compounds such as CR1, LEX-CR1, MCP, DAF, CD59,Factor H, cobra venom factor, FUT-175, complestatin, and K76 COOH.

In some embodiments, the complement inhibitor can be a complementreceptor 2 (CR2)-factor H (FH) molecule comprising: a) a CR2 portioncomprising CR2 (e.g., human CR2) or a fragment thereof, and b) a FHportion comprising a FH or a fragment thereof, wherein the CR2-FHmolecule or fragment thereof is capable of binding to a CR2 ligand, andwherein the CR2-FH molecule is capable of inhibiting complementactivation of the alternative pathway. Exemplary CR2-FH fusion proteinsare described and exemplified in, e.g., International patent applicationpublication nos. WO 2007/149567 and WO 2011/143637, the disclosures ofeach of which are incorporated herein by reference in their entirety. Insome embodiments, the complement inhibitor comprises a targeting domainsuch as CR2 or an anti-C3d antibody as described in, e.g., Internationalpatent application publication no. WO 2011/163412, the disclosure ofwhich is incorporated herein by reference in its entirety. Fusions oftargeting domains with other complement inhibitors such as CD59, CD55,and factor H-like molecules can be used in the methods described hereinas a complement inhibitor. See WO 2011/163412, above.

In some embodiments of any of the methods described herein, theinhibitor of complement is an antagonist antibody or antigen-bindingfragment thereof. The antibody or antigen-binding fragment thereof canbe selected from the group consisting of a humanized antibody, arecombinant antibody, a diabody, a chimerized or chimeric antibody, amonoclonal antibody, a deimmunized antibody, a fully human antibody, asingle chain antibody, an Fv fragment, an Fd fragment, an Fab fragment,an Fab′ fragment, and an F(ab′)₂ fragment.

In some embodiments of any of the methods described herein, theantagonist antibody is an anti-C5 antibody such as eculizumab. In someembodiments, the antagonist antibody is pexelizumab, a C5-bindingfragment of anti-C5 antibody.

In some embodiments of any of the methods described herein, theinhibitor of complement is selected from the group consisting of MB12/22, MB12/22-RGD, ARC187, ARC1905, SSL7, and OmCI.

In some embodiments of any of the methods described herein, the subsetof aHUS-associated biomarker proteins from which a practitioner maydetermine the concentration of one or more (e.g., two, three, four,five, six, seven, eight, nine, 10, or more) of can be: Ba,thrombomodulin, VCAM-1, TNFR1, F1+2, D-dimer, CXCL10, IL-6, clusterin,TIMP-1, FABP-1, β2M, and cystatin C.

In yet another aspect, the disclosure features an array comprising aplurality of binding agents, wherein each binding agent of the pluralityhas a unique address on the array, wherein the array comprises no morethan 500 unique addresses, wherein each binding agent of the pluralitybinds to a different biological analyte protein, and wherein the arraycomprises binding agents that bind to four or more analyte proteins setforth in Table 1, e.g., selected from the group consisting of: aproteolytic fragment of complement component factor B (e.g., Ba or Bb),soluble C5b9 (sC5b9), thrombomodulin, VCAM-1, von Willebrand Factor(vWF), soluble CD40 ligand (sCD40L), prothrombin fragment F1+2, D-dimer,CXCL10, MCP-1, TNFR1, IFN-γ, ICAM-1, IL-1 beta, IL-12 p70, complementcomponent C5a, β2 microglobulin (β2M), clusterin, cystatin C, NAG,TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), CXCL9, KIM-1,IL-18, vascular endothelial cell growth factor (VEGF), IL-6, albumin,IL-8, and CCL5. The array is useful in any of the methods describedherein. In some embodiments, the array is a protein chip. In someembodiments, each address of the array is a well of an assay plate. Insome embodiments, each address of the array is a particle (e.g., a bead)having immobilized thereupon a binding agent.

As used herein, the term “binding agent” includes any naturallyoccurring, synthetic or genetically engineered agent, such as protein,that binds an antigen (e.g., an aHUS biomarker protein). Binding agentscan be or be derived from naturally-occurring antibodies. A bindingprotein or agent can function similarly to an antibody by binding to aspecific antigen to form a complex. Binding agents or proteins caninclude isolated antigen-binding fragments of antibodies.

In some embodiments, the array comprises antibodies that bind to atleast two (e.g., at least three, four, five, six, seven, eight, nine,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) ofthe analyte proteins. For example, the array can comprise bindingagents/antibodies that bind to at least two (e.g., three, four, five,six, seven, eight, nine, 10, 11, 12, or 13) of a proteolytic fragment ofcomplement component factor B (e.g., Ba or Bb), soluble C5b9 (sC5b9),C5a, thrombomodulin, VCAM-1, prothrombin fragment F1+2, D-dimer, sTNFR1,β2 microglobulin (β2M), clusterin, cystatin C, TIMP-1, and fatty acidbinding protein 1 (FABP-1).

In some embodiments, the array comprises no more than 200 (e.g., no morethan 175, 150, 125, 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, or 20)unique addresses.

In yet another aspect, the disclosure features a diagnostic kitcomprising one or more of any of the arrays described herein and,optionally, instructions for (a) obtaining and/or processing abiological sample (e.g., a biological fluid) from a subject and/or (b)measuring one or more analytes in a biological sample (e.g., abiological fluid) from a subject.

In another aspect, the disclosure features a diagnostic kit comprising:(a) an assay plate and (b) at least three binding agents, each bindingagent capable of binding to a different biological analyte, wherein theanalytes are those depicted in Table 1, e.g., selected from the groupconsisting of: a proteolytic fragment of complement component factor B(e.g., Ba or Bb), soluble C5b9 (sC5b9), thrombomodulin, VCAM-1, vonWillebrand Factor (vWF), soluble CD40 ligand (sCD40L), prothrombinfragment F1+2, D-dimer, CXCL10, MCP-1, TNFR1, IFN-γ, ICAM-1, IL-1 beta,IL-12 p70, complement component C5a, β2 microglobulin (β2M), clusterin,cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1),CXCL9, KIM-1, IL-18, vascular endothelial cell growth factor (VEGF),IL-6, albumin, IL-8, and CCL5. In some embodiments, the diagnostic kitcomprises one or more means for measuring the activity of vWF in humanplasma.

In another aspect, the disclosure features a method for diagnosing asubject as having, or being at risk for developing, atypical hemolyticuremic syndrome (aHUS). The method includes: measuring in a biologicalfluid the concentration of at least two aHUS-associated biomarkerproteins selected from the group consisting of: a proteolytic fragmentof complement component factor B (e.g., Ba or Bb), soluble C5b9 (sC5b9),thrombomodulin, VCAM-1, von Willebrand Factor (vWF), soluble CD40 ligand(sCD40L), prothrombin fragment F1+2, D-dimer, CXCL10, MCP-1, TNFR1,IFN-γ, ICAM-1, IL-1 beta, IL-12 p70, complement component C5a, (32microglobulin (β2M), clusterin, cystatin C, NAG, TIMP-1, NGAL, fattyacid binding protein 1 (FABP-1), CXCL9, KIM-1, IL-18, vascularendothelial cell growth factor (VEGF), IL-6, albumin, IL-8, and CCL5.The biological fluid is one obtained from a subject suspected of havingor at risk for developing aHUS. In accordance with the methods, anelevated concentration, as compared to the concentration in a normalcontrol biological fluid of the same type, of at least one of Ba,sC5b-9, C5a, sCD40L, prothrombin fragment F1+2, d-dimer, thrombomodulin,VCAM-1, vWF, FABP-1, β2M, clusterin, cystatin C, TIMP-1, albumin, NGAL,CXCL10, CXCL9, IL-18, TNFR1, VCAM-1, MCP-1, VEGF, CCL5, IL-6, or IFNγ,indicates that the subject has, or is at risk for developing, aHUS. Insome embodiments, the at least two aHUS-associated biomarkers can beselected from Table 11, i.e., at least two (e.g., three, four, five,six, seven, eight, nine, 10, 11, 12, or 13) of a proteolytic fragment ofcomplement component factor B (e.g., Ba or Bb), soluble C5b9 (sC5b9),C5a, thrombomodulin, VCAM-1, prothrombin fragment F1+2, D-dimer, sTNFR1,P2 microglobulin (β2M), clusterin, cystatin C, TIMP-1, and fatty acidbinding protein 1 (FABP-1).

As used herein, the term “normal,” when used to modify the term“individual” or “subject” refers to an individual or group ofindividuals who does/do not have a particular disease or condition(e.g., aHUS) and is also not suspected of having or being at risk fordeveloping the disease or condition. The term “normal” is also usedherein to qualify a biological specimen or sample (e.g., a biologicalfluid) isolated from a normal or healthy individual or subject (or groupof such subjects), for example, a “normal control sample” or “normalcontrol biological fluid”.

In yet another aspect, the disclosure features a method for determiningwhether a patient is experiencing a first acute atypical hemolyticuremic syndrome (aHUS) manifestation. The method comprises: measuringone or both of the concentration of D-dimer (e.g., the plasmaconcentration of d-dimer) and the concentration of fatty acid bindingprotein 1 (FABP-1) (e.g., the urine concentration of FABP-1), wherein anelevation in the d-dimer concentration, relative to the concentration ofd-dimer in a normal control sample, and an elevation in the FABP-1concentration, relative to the concentration of FABP-1 in a normalcontrol sample, indicates that the aHUS patient is experiencing a firstacute aHUS manifestation. In some embodiments, the elevation of one orboth of d-dimer and FABP-1 can be significant elevations.

In another aspect, the disclosure features a method for treatingatypical hemolytic uremic syndrome (aHUS), the method comprisingadministering to a subject having, suspected of having, or at risk fordeveloping, aHUS an inhibitor of complement (e.g., an inhibitor ofcomplement component C5) in an amount and with a frequency sufficient toeffect a physiological change in at least one (e.g., at least two,three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, or 25) aHUS-associated biomarkerproteins, wherein the physiological change is selected from the groupconsisting of: (a) a reduced concentration, as compared to theconcentration in a sample of biological fluid of the same type obtainedfrom the subject prior to treatment with the inhibitor, of at least oneof CXCL10, MCP-1, TNFR1, IFN-γ, a proteolytic fragment of complementcomponent factor B (e.g., Ba or Bb), soluble C5b9 (sC5b9), prothrombinfragment F1+2, d-dimer, thrombomodulin, VCAM-1, von Willebrand Factor(vWF), complement component C5a, sC5b9, P2 microglobulin (β2M),clusterin, cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1(FABP-1), albumin, CXCL10, CXCL9, and KIM-1; or (b) an increasedconcentration, as compared to the concentration in a sample ofbiological fluid of the same type obtained from the subject prior totreatment with the inhibitor, of CCL5. In some embodiments, the at leastone aHUS-associated biomarker can be selected from Table 11, i.e., atleast one (e.g., two, three, four, five, six, seven, eight, nine, 10,11, 12, or 13) of a proteolytic fragment of complement component factorB (e.g., Ba or Bb), soluble C5b9 (sC5b9), C5a, thrombomodulin, VCAM-1,prothrombin fragment F1+2, D-dimer, sTNFR1, P2 microglobulin (β2M),clusterin, cystatin C, TIMP-1, and fatty acid binding protein 1(FABP-1).

In yet another aspect, the disclosure features a method for treatingatypical hemolytic uremic syndrome (aHUS) using a complement inhibitorin a manner sufficient to induce a physiological change in at least twoaHUS-associated biomarker proteins. The method includes: (a) determiningthe concentration of at least two aHUS-associated biomarker proteins ina biological fluid obtained from the subject, wherein theaHUS-associated biomarker proteins are selected from the groupconsisting of: CXCL10, MCP-1, TNFR1, IFN-γ, IL-6, a proteolytic fragmentof complement component factor B (e.g., Ba or Bb), soluble C5b9 (sC5b9),prothrombin fragment F1+2, d-dimer, thrombomodulin, VCAM-1, vonWillebrand Factor (vWF), complement component C5a, P2 microglobulin(β2M), clusterin, cystatin C, NAG, TIMP-1, NGAL, fatty acid bindingprotein 1 (FABP-1), albumin, CXCL9, KIM-1, and CCL5; and (b)administering to a subject having, suspected of having, or at risk fordeveloping, aHUS an inhibitor of complement in an amount and with afrequency sufficient to cause a physiological change in at least each oftwo (2) aHUS-associated biomarker proteins, wherein the physiologicalchange is selected from the group consisting of: (a) a reducedconcentration, as compared to the concentration in a sample ofbiological fluid of the same type obtained from the subject prior totreatment with the inhibitor, of at least one of CXCL10, MCP-1, TNFR1,IFN-γ, IL-6, a proteolytic fragment of complement component factor B(e.g., Ba or Bb), soluble C5b9 (sC5b9), prothrombin fragment F1+2,d-dimer, thrombomodulin, VCAM-1, von Willebrand Factor (vWF), complementcomponent C5a, P2 microglobulin (β2M), clusterin, cystatin C, NAG,TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), albumin, CXCL9, orKIM-1; and (b) an increased concentration in a biological fluid ofobtained from the subject, as compared to the concentration in a sampleof biological fluid of the same type obtained from the subject prior totreatment with the inhibitor, of CCL5. The method can also includedetermining whether the physiological changes occurred. In someembodiments, the at least two aHUS-associated biomarkers can be selectedfrom Table 11, i.e., at least two (e.g., three, four, five, six, seven,eight, nine, 10, 11, 12, or 13) of a proteolytic fragment of complementcomponent factor B (e.g., Ba or Bb), soluble C5b9 (sC5b9), C5a,thrombomodulin, VCAM-1, prothrombin fragment F1+2, D-dimer, sTNFR1, β2microglobulin (β2M), clusterin, cystatin C,TIMP-1, and fatty acidbinding protein 1 (FABP-1).

In some embodiments, the methods can further include the step ofmeasuring the concentrations of at least two individual aHUS-associatedbiomarker proteins in a biological fluid, wherein the aHUS-associatedbiomarker proteins are selected from the group consisting of: aproteolytic fragment of complement component factor B (e.g., Ba or Bb),soluble C5b9 (sC5b9), thrombomodulin, VCAM-1, von Willebrand Factor(vWF), soluble CD40 ligand (sCD40L), prothrombin fragment F1+2, D-dimer,CXCL10, MCP-1, TNFR1, IFN-γ, ICAM-1, IL-1 beta, IL-12 p70, complementcomponent C5a, P2 microglobulin (β2M), clusterin, cystatin C, NAG,TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), CXCL9, KIM-1,IL-18, vascular endothelial cell growth factor (VEGF), IL-6, albumin,IL-8, and CCL5. The biological fluid is obtained from the subject. Insome embodiments, the at least two aHUS-associated biomarkers can beselected from Table 11, i.e., at least two (e.g., three, four, five,six, seven, eight, nine, 10, 11, 12, or 13) of a proteolytic fragment ofcomplement component factor B (e.g., Ba or Bb), soluble C5b9 (sC5b9),C5a, thrombomodulin, VCAM-1, prothrombin fragment F1+2, D-dimer, sTNFR1,β2 microglobulin (β2M), clusterin, cystatin C, TIMP-1, and fatty acidbinding protein 1 (FABP-1).

In some embodiments, any of the methods described herein can includedetermining whether the at least two (e.g., at least three, four, five,six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, or 25) physiological changes have occurred. In someembodiments, the concentrations of at least two of IFN-γ, ICAM-1, IL-1beta, and IL-12 p70 are reduced. In some embodiments, the concentrationsof both Ba and sC5b9 are reduced. In some embodiments, the concentration(e.g., the urine concentration) of each of C5a and sC5b9 is reduced. Insome embodiments of any of the methods described herein, theconcentrations (e.g., the urine concentration) of at least two (e.g., atleast three, four, five, six, or all) of β2M, clusterin, cystatin C,NAG, TIMP-1, NGAL, and FABP-1 are reduced. In some embodiments, theconcentrations (e.g., the urine concentration) of CXCL10, CXCL9, and/orKIM-1 are reduced. In some embodiments, the concentrations (e.g., plasmaconcentration) of one or both of D-dimer and F1+2 are reduced. In someembodiments, the concentrations (e.g., the serum and/or plasmaconcentrations) of at least two (e.g., at least three, or all) ofsCD40L, prothrombin fragment F1+2, and D-dimer are reduced. In someembodiments, the concentrations of thrombomodulin, VCAM-1, and/or vWFare reduced. In some embodiments, the concentrations (e.g., the serumconcentrations) of CXCL10, MCP-1, and TNFR1 are reduced. In someembodiments, the concentrations (e.g., the serum concentrations) of atleast two (e.g., at least three, four, or all) of IFN-γ, ICAM-1, IL-1beta, and IL-12 p70 are reduced. In some embodiments, the at least twophysiological changes can be a reduction in concentration of at leasttwo aHUS-associated biomarkers selected from Table 11, i.e., at leasttwo (e.g., three, four, five, six, seven, eight, nine, 10, 11, 12, or13) of a proteolytic fragment of complement component factor B (e.g., Baor Bb), soluble C5b9 (sC5b9), C5a, thrombomodulin, VCAM-1, prothrombinfragment F1+2, D-dimer, sTNFR1, β2 microglobulin (β2M), clusterin,cystatin C, TIMP-1, and fatty acid binding protein 1 (FABP-1).

In some embodiments of any of the methods described herein, the Baconcentration (e.g., plasma Ba concentration) is reduced by at least 10%by week 6 post-initiation of treatment. In some embodiments of any ofthe methods described herein, the Ba concentration (e.g., plasma Baconcentration) is reduced by at least 30% by week 12 post-initiation oftreatment. In some embodiments of any of the methods described herein,the C5a concentration (e.g., urinary C5a concentration) is reduced by atleast 40% by week 3 post-initiation of treatment. In some embodiments ofany of the methods described herein, the C5a concentration (e.g.,urinary C5a concentration) is reduced by at least 70% by week 6post-initiation of treatment. In some embodiments of any of the methodsdescribed herein, the C5b-9 concentration (e.g., urinary or plasma C5b-9concentration) is reduced by at least 50% by week 3 post-initiation oftreatment. In some embodiments of any of the methods described herein,the F1+2 concentration (e.g., the plasma concentration of F1+2) isreduced by at least 20% by week 6 post-initiation of treatment. In someembodiments of any of the methods described herein, the d-dimerconcentration (e.g., the plasma concentration of d-dimer) is reduced byat least 40% by week 6 post-initiation of treatment. In some embodimentsof any of the methods described herein, the thrombomodulin concentration(e.g., the serum concentration of thrombomodulin) is reduced by at least20% by week 12 post-initiation of treatment. In some embodiments of anyof the methods described herein, the VCAM-1 concentration (e.g., theserum concentration of VCAM-1) is reduced by at least 20% by week 12post-initiation of treatment.

In some embodiments of any of the methods described herein, theinhibitor of complement is administered to the subject in an amount andwith a frequency sufficient to effect a physiological change in three ormore aHUS-associated biomarkers. In some embodiments, the inhibitor ofcomplement is administered to the subject in an amount and with afrequency sufficient to effect a physiological change in at least fouraHUS-associated biomarkers. In some embodiments, the inhibitor ofcomplement is administered to the subject in an amount and with afrequency sufficient to effect a physiological change in at least fiveaHUS-associated biomarkers. In some embodiments, the inhibitor ofcomplement is administered to the subject in an amount and with afrequency sufficient to effect a physiological change in at least 10aHUS-associated biomarkers. In some embodiments, the inhibitor ofcomplement component C5 is administered to the subject in an amount andwith a frequency sufficient to effect a physiological change in 15 ormore aHUS-associated biomarkers.

In some embodiments, a physiological change in at least two (e.g., atleast three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more) aHUS-associatedbiomarker proteins occurs within two days, three days, four days, fivedays, six days, one week, two weeks, three weeks, four weeks, six weeks,two months, nine weeks, or three months or more after administration(e.g., chronic administration) of the inhibitor.

In some embodiments, the concentration of at least one (e.g., at leasttwo, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) aHUS-associated biomarkerprotein is reduced by at least 5 (e.g., at least 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, or 70) % following administration of theinhibitor.

In some embodiments, the concentration of at least one (e.g., at leasttwo, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) aHUS-associated biomarkerprotein is reduced to within 50 (e.g., 49, 48, 47, 46, 45, 44, 43, 42,41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24,23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5,4, 3, 2, or 1) % of the normal concentration of the biomarker proteinfollowing administration of one or more doses of the inhibitor.

In some embodiments of any of the methods described herein, theconcentration of FABP-1 (e.g., urinary FABP-1) is reduced by at least80% (e.g., 85, 90, 95, or up to 100%) following administration of aninhibitor of human complement (e.g., an anti-C5 antibody). In someembodiments of any of the methods described herein, the concentration ofcystatin-C (e.g., urinary cystatin-C) is reduced by at least 80% (e.g.,85, 90, 95, 99, or up to 100%) following administration of an inhibitorof human complement (e.g., an anti-C5 antibody). In some embodiments ofany of the methods described herein, the concentration of clusterin(e.g., urinary clusterin) is reduced by at least 80% (e.g., 85, 90, 95,98, or up to 100%) following administration of an inhibitor of humancomplement (e.g., an anti-C5 antibody). In some embodiments of any ofthe methods described herein, the concentration of a proteolyticfragment of factor B (e.g., Ba) is reduced by at least 10% (e.g., 15,20, 25, 30, or 40%) following administration of an inhibitor of humancomplement (e.g., an anti-C5 antibody). In some embodiments of any ofthe methods described herein, the concentration of sTNFR1 is reduced byat least 80% (e.g., 85, 90, or more %) following administration of aninhibitor of human complement (e.g., an anti-C5 antibody). In someembodiments of any of the methods described herein, the concentration ofthrombomodulin or sVCAM-1 is reduced by at least 80% (e.g., 85, 90, 95,or up to 100%) following administration of an inhibitor of humancomplement (e.g., an anti-C5 antibody). In some embodiments of any ofthe methods described herein, the concentration of one or both of F1+2or D-dimer is reduced by at least 80% (e.g., 85, 90, 95, or more %)following administration of an inhibitor of human complement (e.g., ananti-C5 antibody).

In some embodiments of any of the methods described herein, theconcentration of at least one (e.g., at least two, three, four, five,six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, or 25) of the aHUS-associated biomarker proteins isnormalized following administration of the inhibitor. In someembodiments, the concentrations (e.g., the urine concentrations) of atleast three of (32 microglobulin (β2M), clusterin, cystatin C, NAG,TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), CXCL10, CXCL9, andKIM-1 are normalized.

As used herein, the term “normalized” or like grammatical terms, whenused in the context of the effect of a complement inhibitor therapy onthe concentration or activity of an aHUS biomarker protein, refers to aconcentration or activity measured in a biological fluid of a biomarkerprotein that has been brought within 50 (e.g., 49, 48, 47, 46, 45, 44,43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26,25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7,6, 5, 4, 3, 2, or 1) % of the average concentration or activity range ofthe aHUS biomarker protein as measured in a sample of the same type ofbiological fluid obtained from a group of healthy individuals (normalindividuals). For example, treatment of an aHUS patient with acomplement inhibitor can normalize an elevated urine clusterinconcentration to within, e.g., 20% of the normal average urineconcentration range of clusterin. In some embodiments, treatment withthe complement inhibitor would restore the urine concentration ofclusterin to within the normal average urine concentration range ofclusterin.

In some embodiments of any of the methods described herein, the subjecthas received dialysis at least once (e.g., at least twice, thrice, fourtimes, or five times or more) within the three months (e.g., 11, 10, 9,8, 7, 6, 5, 4, 3, 2, or 1 week(s)) prior to treatment with theinhibitor. For example, in some embodiments the subject receiveddialysis one time two months before receiving the complement inhibitortherapy. In another example, the subject may be one who has receiveddialysis three times within the three month period just prior toreceiving the complement inhibitor therapy. In some embodiments of anyof the methods described herein, relative to the concentration in ahealthy subject, the concentrations of one or more of TNFR1, Ba,thrombomodulin fragment F1+2, and sC5b9 are elevated. In someembodiments of any of the methods described herein, relative to theconcentrations (e.g., the urine concentrations) in a healthy human, theconcentrations of one or more of β2M, sC5b9, C5a, cystatin C, clusterin,TIMP-1, and NGAL are elevated.

In some embodiments of any of the methods described herein, the subject(e.g., a human subject) is experiencing a first acute aHUSmanifestation. For example, prior to treatment with the complementinhibitor, the subject can have elevated concentrations, relative to thenormal concentrations, of one or both of D-dimer and FABP-1.

In some embodiments of any of the methods described herein, the subject(e.g., a human subject) is one having aHUS, but deemed to be in clinicalremission (e.g., the subject is one having normal levels of platelets orother hematologic markers such as LDH or haptoglobin). In someembodiments, such a subject is one having elevated levels of one or moreof the aHUS biomarkers described herein including, but not limited, oneor more of Ba, D-dimer, VCAM-1, and prothrombin fragments 1+2.

It is understood that for any of the methods described herein, theconcentration and/or activity of one or more aHUS biomarker proteins canbe determined. For example, in some embodiments, a practitioner maymeasure the activity of vWF in a biological sample obtained from thesubject as a proxy for the concentration of vWF (or other biomarkerproteins) in the sample. Methods for assessing relative activity of theaHUS biomarker proteins set forth in Table 1 are known in the art.

As discussed in detail herein (for example, in the working examples),aHUS is a genetic, life threatening disease involving chronic complementdysregulation. Patients afflicted with the disease suffer from, amongother things, thrombotic microangiopathy (TMA), which can result instroke and kidney failure. Eculizumab, an antagonist anti-C5 antibody,has been shown to dramatically reduce TMA, normalize platelet levels,and improve renal function of aHUS patients. Yet, even with the clearand robust clinical benefit of complement inhibitor therapy for aHUSpatients, some patients still experience elevated levels of several aHUSbiomarker proteins in the face of treatment. For example, the inventorshave discovered that, in some patients, a proteolytic fragment ofcomplement component factor B (e.g., Ba or Bb) levels (e.g., in plasma)do not normalize following treatment with an antagonist anti-C5antibody. In addition, for some patients, levels of prothrombin fragment1+2, D-dimer, thrombomodulin, VCAM-1, TNFR1, and CXCL10 levels arereduced but do not normalize over time. While the disclosure is notbound by any particular theory or mechanism of action, theseobservations suggest that, for some patients, low levels of inflammationand coagulopathy may persist even with complement inhibitor therapy.Thus, the disclosure contemplates methods in which a complementinhibitor is administered in combination with a second therapy toaddress the low level of persistent inflammation in some patients withaHUS.

Thus, in yet another aspect, the disclosure features a method fortreating atypical hemolytic uremic syndrome (aHUS). The method comprisesadministering (e.g., chronically administering) to a subject (e.g., ahuman subject) having, suspected of having, or at risk for developing,aHUS a therapeutically effective amount of an inhibitor of complement(e.g., an inhibitor of complement component C5) and a therapeuticallyeffective amount of: (i) an anti-coagulant, (ii) a fibrinolytic agent;(iii) an anti-inflammatory agent; or (iv) an inhibitor of IL-6, IL-8,CXCL-9, IL-18, or VEGF. In some embodiments, two inhibitors ofcomplement can be used (e.g., an inhibitor of C5 and an inhibitor of C3,such as, an anti-Factor B antibody, an anti-C3 antibody, or an anti-C3bantibody). In some embodiments, at the time of discontinuing therapywith an inhibitor of C5, an inhibitor of complement component C3 can beadministered to the patient for a time sufficient to reduce upstreamalternative pathway activation.

In some embodiments, the methods can include monitoring the status ofone or more aHUS biomarkers and determining whether to start a secondtherapy (in addition to complement inhibitor therapy) or modify thedosing regimen of one or more second therapies being administered to anaHUS patient. For example, during treatment (e.g., chronic treatment)with a complement inhibitor, the concentration of one or more aHUSassociated biomarker proteins can be measured in one or more biologicalfluids obtained from the subject. If the concentration of one or more ofthe biomarker proteins has not normalized and/or remains elevated, amedical practitioner may elect to administer to the subject one or moreadditional secondary agents (e.g., anti-inflammatories) to address anypathophysiological effects resulting from the elevated biomarkers.

The complement inhibitor can be any of those described herein. In someembodiments of any of the methods described herein, the inhibitor isantibody or an antigen binding fragment thereof, a small molecule, apolypeptide, a polypeptide analog, a peptidomimetic, or an aptamer. Insome embodiments, the inhibitor can be one that inhibits one or more ofcomplement components C1, C2, C3, C4, C5, C6, C7, C8, C9, Factor D,Factor B, properdin, MBL, MASP-1, MASP-2, or biologically activefragments of any of the foregoing. In some embodiments of any of themethods described herein, the complement inhibitor inhibits one or bothof the generation of the anaphylatoxic activity associated with C5aand/or the assembly of the membrane attack complex associated with C5b.

The compositions can also contain naturally occurring or soluble formsof complement inhibitory compounds such as CR1, LEX-CR1, MCP, DAF, CD59,Factor H, cobra venom factor, FUT-175, complestatin, and K76 COOH.

In some embodiments of any of the methods described herein, theinhibitor of complement is an antagonist antibody or antigen-bindingfragment thereof. The antibody or antigen-binding fragment thereof canbe selected from the group consisting of a humanized antibody, arecombinant antibody, a diabody, a chimerized or chimeric antibody, amonoclonal antibody, a deimmunized antibody, a fully human antibody, asingle chain antibody, an Fv fragment, an Fd fragment, an Fab fragment,an Fab′ fragment, and an F(ab′)₂ fragment.

In some embodiments of any of the methods described herein, theantagonist antibody is an anti-C5 antibody such as eculizumab. In someembodiments, the antagonist antibody is pexelizumab, a C5-bindingfragment of anti-C5 antibody.

In some embodiments of any of the methods described herein, theinhibitor of complement is selected from the group consisting ofMB12/22, MB12/22-RGD, ARC187, ARC1905, SSL7, and OmCI.

In some embodiments, the anti-coagulant is selected from the groupconsisting of: a coumarin, heparin, a factor Xa inhibitor, and athrombin inhibitor. Examples of anti-coagulants include, e.g., warfarin(Coumadin), aspirin, heparin, phenindione, fondaparinux, idraparinux,and thrombin inhibitors (e.g., argatroban, lepirudin, bivalirudin, ordabigatran).

In some embodiments, the fibrinolytic agent is selected from the groupconsisting of ancrod, ε-aminocaproic acid, antiplasmin-a₁, prostacyclin,and defibrotide.

In some embodiments, the anti-inflammatory agent is an anti-cytokineagent such as an antagonist antibody (or antigen-binding fragmentthereof) or a soluble cytokine receptor, which binds to an inflammatorycytokine and inhibits the activity of the cytokine. The anti-cytokineagent can be, e.g., a TNF inhibitor (e.g., an anti-TNF antibody orsoluble TNF receptor protein) or an anti-CD20 agent.

Anti-inflammatory agents also include, e.g., steroids (e.g.,dexamethasone), non-steroidal anti-inflammatory drugs (NSAIDs) (e.g.,indomethacin, naproxen, sulindac, diclofenac, aspirin, flurbiprofen,oxaprozin, salsalate, difunisal, piroxicam, etodolac, meclofenamate,ibuprofen, fenoprofen, ketoprofen, nabumetone, tolmetin, cholinemagnesium salicylate, COX-2 inhibitors, TNF alpha antagonists(etanercept, adalimumab, infliximab, golimumab), disease modifyinganti-rheumatic drugs (DMARDS) (e.g., sulfasalazine, methotrexate),cyclosporin, retinoids and corticosteroids.

In yet another aspect, the disclosure features a method for determiningwhether the concentration of one or more aHUS-associated biomarkerproteins are elevated in a patient having, suspected of having, or atrisk for developing, atypical hemolytic uremic syndrome (aHUS), whereinthe method comprises: (i) measuring in a biological sample obtained fromthe patient the concentration of each of at least two aHUS-associatedbiomarkers from Table 11 (infra), i.e., selected from the groupconsisting of: a proteolytic fragment of factor B, C5a, soluble C5b-9(sC5b-9), soluble TNFR1 (sTNFR1), soluble VCAM-1 (sVCAM-1),thrombomodulin, prothrombin fragments 1 and 2 (F1+2), D-dimer,clusterin, TIMP-1, FABP-1, beta-2 microglobulin (β2m), and cystatin-C,and (ii) determining whether the patient has an elevated concentrationof each of at least two of the aHUS-associated biomarkers as compared toa normal control concentration of the same at least two biomarkers. Insome embodiments, the at least two aHUS-associated biomarker proteinsare measured using an immunoassay, such as, an enzyme-linkedimmunosorbent assay (ELISA) or a radioimmunoassay (RIA). The biologicalfluid can be, e.g., blood, a blood fraction (e.g., plasma or serum), orurine. It is understood that any combination of any two or more (e.g.,three, four, five, six, seven, eight, nine, 10, 11 or 12) of theaforementioned aHUS-biomarkers can be measured and analyzed inaccordance with the methods described herein.

In another aspect, the disclosure features a method for diagnosing apatient as having atypical hemolytic uremic syndrome (aHUS) (orconfirming a diagnosis of aHUS, e.g., where the patient has met two ormore of the inclusion criteria discussed under Example 1), wherein themethod comprises: (i) measuring in a biological sample obtained from apatient suspected of having aHUS or at risk of developing aHUS theconcentration of each of at least two aHUS-associated biomarkersselected from the group consisting of: a proteolytic fragment of factorB, C5a, soluble C5b-9 (sC5b-9), soluble TNFR1 (sTNFR1), soluble VCAM-1(sVCAM-1), thrombomodulin, prothrombin fragments 1 and 2 (F1+2),D-dimer, clusterin, TIMP-1, FABP-1, beta-2 microglobulin (β2m), andcystatin-C, and (ii) diagnosing a patient as having aHUS (or confirminga diagnosis of aHUS) if the concentration of each of at least two of theaHUS-associated biomarkers are elevated as compared to a normal controlconcentration of the same at least two biomarkers. In some embodiments,the at least two aHUS-associated biomarker proteins are measured usingan immunoassay, such as, an enzyme-linked immunosorbent assay (ELISA) ora radioimmunoassay (RIA). The biological fluid can be, e.g., blood, ablood fraction (e.g., plasma or serum), or urine. It is understood thatany combination of any two or more (e.g., three, four, five, six, seven,eight, nine, 10, 11 or 12) of the aforementioned aHUS-biomarkers can bemeasured and analyzed in accordance with the methods described herein.

A normal control concentration, as used in any of the methods describedherein, can be (or can be based on), e.g., the concentration of a givenaHUS-associated biomarker protein in a biological sample or biologicalsamples obtained from one or more (e.g., two, three, four, five, six,seven, eight, nine, 10, 15, 20, 25, 30, 35, or 40 or more) healthyindividuals. In some embodiments, a normal control concentration of abiomarker can be (or can be based on), e.g., the concentration of thebiomarker in a pooled sample obtained from two or more (e.g., two,three, four, five, six, seven, eight, nine, 10, 15, 20, 25, 30, 35, or40 or more) healthy individuals. In some embodiments of any of themethods described herein, the pooled samples can be from healthyindividuals or, at least, individuals who do not have or are notsuspected of having (nor at risk for developing) aHUS. For example,determining whether a subject is one having aHUS can involve comparingthe measured concentration of one or more complement component proteins(e.g., Table 1 or Table 11) in a biological sample (or several differenttypes of biological samples) obtained from the patient and comparing themeasured concentration to the average concentration of the same proteinsin the pooled healthy samples. Such healthy human control concentrationscan be, in some embodiments, a range of values, or a median or meanvalue obtained from the range.

In some embodiments, the concentration of at least one aHUS-associatedbiomarker is measured in two or more types of biological fluid. In someembodiments, the concentration of the first of the at least two aHUSbiomarker proteins is measured in one type of biological fluid and theconcentration of the second of the at least two aHUS biomarker proteinsis measured in a second type of fluid.

In some embodiments of any of the methods described herein, theconcentration of the proteolytic fragment of factor B is measured. Thefragment can be, e.g., Ba. The biological sample can be a plasma sample.As described in Table 11, the normal control concentration of Ba can beless than 1000 ng/mL. The normal control concentration of Ba can be lessthan 600 ng/mL. The normal control concentration of Ba can be between300 and 600 ng/mL.

In some embodiments, the concentration of Ba in the biological sample isdeemed elevated when it is at least two fold greater than the normalcontrol concentration of Ba. In some embodiments, the concentration ofBa in the biological sample is deemed elevated when it is at least fivefold greater than the normal control concentration of Ba. In someembodiments, the concentration of Ba in the biological sample is deemedelevated when it is at least 1500 ng/mL. In some embodiments, theconcentration of Ba in the biological sample is deemed elevated when itis at least 2500 ng/mL.

In some embodiments of any of the methods described herein, theconcentration of C5a is measured. The biological sample in which C5a ismeasured can be a urine sample. And in some embodiments, the normalcontrol concentration of C5a is less than 2 ng per mg of urinarycreatinine. In some embodiments, the normal control concentration of C5ais less than 1 ng per mg of urinary creatinine. In some embodiments, thenormal control concentration of C5a is between 0 and 0.7 ng per mg ofurinary creatinine.

In some embodiments of any of the methods described herein, theconcentration of C5a in the biological sample is deemed elevated when itis at least two fold greater than the normal control concentration ofC5a. In some embodiments, the concentration of C5a in the biologicalsample is deemed elevated when it is at least ten fold greater than thenormal control concentration of C5a. In some embodiments, theconcentration of C5a in the biological sample is deemed elevated when itis at least forty fold greater than the normal control concentration ofC5a. In some embodiments, the concentration of C5a in the biologicalsample is deemed elevated when it is at least 5 ng per mg of urinarycreatinine. In some embodiments, the concentration of C5a in thebiological sample is deemed elevated when it is at least 9 ng per mg ofurinary creatinine.

In some embodiments of any of the methods described herein, theconcentration of sC5b-9 is measured. The biological sample in whichsC5b-9 is measured can be a urine sample. And in some embodiments, thenormal control concentration of sC5b-9 is less than 2 ng per mg ofurinary creatinine. The normal control concentration of sC5b-9 can beless than 1 ng per mg of urinary creatinine. In some embodiments, thenormal control concentration of sC5b-9 is between 0 and 0.6 ng per mg ofurinary creatinine.

In some embodiments of any of the methods described herein, theconcentration of sC5b-9 in the biological sample is deemed elevated whenit is at least ten fold greater than the normal control concentration ofsC5b-9. In some embodiments, the concentration of sC5b-9 in thebiological sample is deemed elevated when it is at least fifty foldgreater than the normal control concentration of sC5b-9. In someembodiments, the concentration of sC5b-9 in the biological sample isdeemed elevated when it is at least one hundred fold greater than thenormal control concentration of sC5b-9. In some embodiments, theconcentration of sC5b-9 in the biological sample is deemed elevated whenit is at least 20 ng per mg of urinary creatinine. In some embodiments,the concentration of sC5b-9 in the biological sample is deemed elevatedwhen it is at least 30 ng per mg of urinary creatinine.

In some embodiments of any of the methods described herein, theconcentration of sTNFR1 is measured. The biological sample in whichsTNFR1 is measured can be a serum sample. And in some embodiments, thenormal control concentration of sTNFR1 is less than 2000 pg/mL. In someembodiments, the normal control concentration of sTNFR1 is less than1500 pg/mL. In some embodiments, the normal control concentration ofsTNFR1 is between 400 and 1500 pg/mL.

In some embodiments of any of the methods described herein, theconcentration of sTNFR1 in the biological sample is deemed elevated whenit is at least two fold greater than the normal control concentration ofsTNFR1. In some embodiments, the concentration of sTNFR1 in thebiological sample is deemed elevated when it is at least five foldgreater than the normal control concentration of sTNFR1. In someembodiments, the concentration of sTNFR1 in the biological sample isdeemed elevated when it is at least fifteen fold greater than the normalcontrol concentration of sTNFR1. In some embodiments, the concentrationof sTNFR1 in the biological sample is deemed elevated when it is atleast 10,000 pg/mL. In some embodiments, the concentration of sTNFR1 inthe biological sample is deemed elevated when it is at least 15,000pg/mL.

In some embodiments of any of the methods described herein, theconcentration of sVCAM-1 is measured. The biological sample in whichsVCAM-1 is measured can be a serum sample. And in some embodiments, thenormal control concentration of sVCAM-1 is less than 500 ng/mL. In someembodiments, the normal control concentration of sVCAM-1 is less than300 ng/mL. In some embodiments, the normal control concentration ofsVCAM-1 is between 100 and 500 ng/mL.

In some embodiments of any of the methods described herein, theconcentration of sVCAM-1 in the biological sample is deemed elevatedwhen it is at least 10% greater than the normal control concentration ofsVCAM-1. In some embodiments, the concentration of sVCAM-1 in thebiological sample is deemed elevated when it is at least 30% greaterthan the normal control concentration of sVCAM-1. In some embodiments,the concentration of sVCAM-1 in the biological sample is deemed elevatedwhen it is at least 50% greater than the normal control concentration ofsVCAM-1. In some embodiments, the concentration of sVCAM-1 in thebiological sample is deemed elevated when it is at least 550 ng/mL. Insome embodiments, the concentration of sVCAM-1 in the biological sampleis deemed elevated when it is at least 650 ng/mL.

In some embodiments of any of the methods described herein, theconcentration of thrombomodulin is measured. The biological sample inwhich thrombomodulin is measured can be a plasma sample. And in someembodiments, the normal control concentration of thrombomodulin is lessthan 5 ng/mL. In some embodiments, the normal control concentration ofthrombomodulin is less than 3 ng/mL. In some embodiments, the normalcontrol concentration of thrombomodulin is between 2 and 6 ng/mL.

In some embodiments of any of the methods described herein, theconcentration of thrombomodulin in the biological sample is deemedelevated when it is at least 10% greater than the normal controlconcentration of thrombomodulin. In some embodiments, the concentrationof thrombomodulin in the biological sample is deemed elevated when it isat least 30% greater than the normal control concentration ofthrombomodulin. In some embodiments, the concentration of thrombomodulinin the biological sample is deemed elevated when it is at least 50%greater than the normal control concentration of thrombomodulin. In someembodiments, the concentration of thrombomodulin in the biologicalsample is deemed elevated when it is at least 8 ng/mL. In someembodiments, the concentration of thrombomodulin in the biologicalsample is deemed elevated when it is at least 10 ng/mL.

In some embodiments of any of the methods described herein, theconcentration of F1+2 is measured. The biological sample in which F1+2is measured can be a plasma sample. And in some embodiments, the normalcontrol concentration of F1+2 is less than 400 pmol/L. In someembodiments, the normal control concentration of F1+2 is less than 300pmol/L. In some embodiments, the normal control concentration of F1+2 isbetween 50 and 400 pmol/L.

In some embodiments of any of the methods described herein, theconcentration of F1+2 in the biological sample is deemed elevated whenit is at least 30% greater than the normal control concentration ofF1+2. In some embodiments, the concentration of F1+2 in the biologicalsample is deemed elevated when it is at least 50% greater than thenormal control concentration of F1+2. In some embodiments, theconcentration of F1+2 in the biological sample is deemed elevated whenit is at least 100% greater than the normal control concentration ofF1+2. In some embodiments, the concentration of F1+2 in the biologicalsample is deemed elevated when it is at least 900 pmol/L. In someembodiments, the concentration of F1+2 in the biological sample isdeemed elevated when it is at least 1000 pmol/L.

In some embodiments of any of the methods described herein, theconcentration of D-dimer is measured. The biological sample in whichD-dimer is measured can be a plasma sample. And in some embodiments, thenormal control concentration of D-dimer is less than 500 μg/L. In someembodiments, the normal control concentration of D-dimer is less than400 μg/L. In some embodiments, the normal control concentration ofD-dimer is between 100 and 500 μg/L.

In some embodiments of any of the methods described herein, theconcentration of D-dimer in the biological sample is deemed elevatedwhen it is at least two-fold greater than the normal controlconcentration of D-dimer. In some embodiments, the concentration ofD-dimer in the biological sample is deemed elevated when it is at leastfive-fold greater than the normal control concentration of D-dimer. Insome embodiments, the concentration of D-dimer in the biological sampleis deemed elevated when it is at least ten-fold greater than the normalcontrol concentration of D-dimer. In some embodiments, the concentrationof D-dimer in the biological sample is deemed elevated when it is atleast 1500 μg/L. In some embodiments, the concentration of D-dimer inthe biological sample is deemed elevated when it is at least 2500 μg/L.

In some embodiments of any of the methods described herein, theconcentration of clusterin is measured. The biological sample in whichclusterin is measured can be a urine sample. And in some embodiments,the normal control concentration of clusterin is less than 500 ng per mgof urinary creatinine. The normal control concentration of clusterin canbe, e.g., less than 400 ng per mg of urinary creatinine. In someembodiments, the normal control concentration of clusterin is between 0and 500 ng per mg of urinary creatinine.

In some embodiments of any of the methods described herein, theconcentration of clusterin in the biological sample is deemed elevatedwhen it is at least two-fold greater than the normal controlconcentration of clusterin. In some embodiments, the concentration ofclusterin in the biological sample is deemed elevated when it is atleast five-fold greater than the normal control concentration ofclusterin. In some embodiments, the concentration of clusterin in thebiological sample is deemed elevated when it is at least ten-foldgreater than the normal control concentration of clusterin. In someembodiments, the concentration of clusterin in the biological sample isdeemed elevated when it is at least 900 ng per mg of urinary creatinine.In some embodiments, the concentration of clusterin in the biologicalsample is deemed elevated when it is at least 1200 ng per mg of urinarycreatinine.

In some embodiments of any of the methods described herein, theconcentration of TIMP-1 is measured. The biological sample in whichTIMP-1 is measured can be a urine sample. And in some embodiments, thenormal control concentration of TIMP-1 is less than 10 ng per mg ofurinary creatinine. In some embodiments, the normal controlconcentration of TIMP-1 is less than 5 ng per mg of urinary creatinine.In some embodiments, the normal control concentration of TIMP-1 isbetween 0 and 10 ng per mg of urinary creatinine.

In some embodiments of any of the methods described herein, theconcentration of TIMP-1 in the biological sample is deemed elevated whenit is at least two-fold greater than the normal control concentration ofTIMP-1. In some embodiments, the concentration of TIMP-1 in thebiological sample is deemed elevated when it is at least ten-foldgreater than the normal control concentration of TIMP-1. In someembodiments, the concentration of TIMP-1 in the biological sample isdeemed elevated when it is at least twenty-fold greater than the normalcontrol concentration of TIMP-1. In some embodiments, the concentrationof TIMP-1 in the biological sample is deemed elevated when it is atleast 15 ng per mg of urinary creatinine. In some embodiments, theconcentration of TIMP-1 in the biological sample is deemed elevated whenit is at least 20 ng per mg of urinary creatinine.

In some embodiments of any of the methods described herein, theconcentration of FABP-1 (also referred to herein as L-FABP-1) ismeasured. The biological sample in which C5a is measured can be a urinesample. And in some embodiments, the normal control concentration ofFABP-1 is less than 20 ng per mg of urinary creatinine. In someembodiments, the normal control concentration of FABP-1 is less than 15ng per mg of urinary creatinine. In some embodiments, the normal controlconcentration of FABP-1 is between 0 and 20 ng per mg of urinarycreatinine.

In some embodiments of any of the methods described herein, theconcentration of FABP-1 in the biological sample is deemed elevated whenit is at least two-fold greater than the normal control concentration ofFABP-1. In some embodiments, the concentration of FABP-1 in thebiological sample is deemed elevated when it is at least ten-foldgreater than the normal control concentration of FABP-1. In someembodiments, the concentration of FABP-1 in the biological sample isdeemed elevated when it is at least twenty-fold greater than the normalcontrol concentration of FABP-1. In some embodiments, the concentrationof FABP-1 in the biological sample is deemed elevated when it is atleast 40 ng per mg of urinary creatinine. In some embodiments, theconcentration of FABP-1 in the biological sample is deemed elevated whenit is at least 50 ng per mg of urinary creatinine.

In some embodiments of any of the methods described herein, theconcentration of β2m is measured. The biological sample in which β2m ismeasured can be a urine sample. And in some embodiments, the normalcontrol concentration of β2m is less than 5 μg per mg of urinarycreatinine. In some embodiments, the normal control concentration of β2mis less than 3 μg per mg of urinary creatinine. In some embodiments, thenormal control concentration of β2m is between 0 and 5 μg per mg ofurinary creatinine.

In some embodiments of any of the methods described herein, theconcentration of β2m in the biological sample is deemed elevated when itis at least two-fold greater than the normal control concentration ofβ2m. In some embodiments of any of the methods described herein, theconcentration of β2m in the biological sample is deemed elevated when itis at least ten-fold greater than the normal control concentration ofβ2m. In some embodiments, the concentration of β2m in the biologicalsample is deemed elevated when it is at least twenty-fold greater thanthe normal control concentration of β2m. In some embodiments, theconcentration of β2m in the biological sample is deemed elevated when itis at least 15 μg per mg of urinary creatinine. In some embodiments, theconcentration of β2m in the biological sample is deemed elevated when itis at least 20 μg per mg of urinary creatinine.

In some embodiments of any of the methods described herein, theconcentration of cystatin-C is measured. The biological sample in whichcystatin-C is measured can be a urine sample. And in some embodiments,the normal control concentration of cystatin-C is less than 400 ng permg of urinary creatinine. In some embodiments, the normal controlconcentration of cystatin-C is less than 300 ng per mg of urinarycreatinine. In some embodiments, the normal control concentration ofcystatin-C is between 0 and 400 ng per mg of urinary creatinine.

In some embodiments of any of the methods described herein, theconcentration of cystatin-C in the biological sample is deemed elevatedwhen it is at least two-fold greater than the normal controlconcentration of cystatin-C. In some embodiments, the concentration ofcystatin-C in the biological sample is deemed elevated when it is atleast ten-fold greater than the normal control concentration ofcystatin-C. In some embodiments, the concentration of cystatin-C in thebiological sample is deemed elevated when it is at least twenty-foldgreater than the normal control concentration of cystatin-C. In someembodiments, the concentration of cystatin-C in the biological sample isdeemed elevated when it is at least 900 ng per mg of urinary creatinine.In some embodiments, the concentration of cystatin-C in the biologicalsample is deemed elevated when it is at least 1200 ng per mg of urinarycreatinine.

In some embodiments of any of the methods described herein, theconcentrations of two or more of proteolytic fragments of factor B, C5a,and sC5b-9 are measured. In some embodiments of any of the methodsdescribed herein, the concentrations of C5a and sC5b-9 are measured. Insome embodiments of any of the methods described herein, theconcentrations of sVCAM-1 and thrombomodulin are measured. In someembodiments of any of the methods described herein, the concentrationsof F1+2 and D-dimer are measured. In some embodiments of any of themethods described herein, the concentrations of two or more ofclusterin, TIMP-1, β2m, FABP-1, and cystatin-C are measured.

In yet another aspect, the disclosure features a method for assessingthe level of alternative pathway activation in a patient having aHUS,suspected of having aHUS, or at risk for developing aHUS, before,during, or after treatment with a complement inhibitor, such as, ananti-C5 antibody. The method comprises: measuring the concentration of aproteolytic fragment of factor B (e.g., Ba or Bb) in a biological sampleobtained from a patient treated with an inhibitor of complement (e.g.,an inhibitor of human complement component C5, such as, an anti-C5antibody).

In yet another aspect, the disclosure features a method for determiningwhether a patient has responded to therapy with a complement inhibitor(e.g., had a reduction in risk of developing thrombosis or had areduction in the number, frequency, or occurrence of thromboticmicroangiopathy), the method comprising measuring the concentration ofone or more biomarkers of thrombosis or coagulation set forth in Table 1or 11, e.g., F1+2, D-dimer, vWF, or thrombomodulin, in a biologicalsample obtained from a patient at elevated risk of, suffering from, orsuspected of having, thrombotic microangiopathy (TMA) and treated with acomplement inhibitor; and determining that the patient has responded tothe therapy if the concentration of the one or more biomarkers in thebiological sample is reduced, as compared to the concentration of theone or more biomarkers in a biological sample of the same type obtainedfrom the patient prior to treatment with the complement inhibitor ordetermining that the patient has not responded to the therapy if theconcentration of the one or more biomarkers in the biological sample isnot reduced, as compared to the concentration of the one or morebiomarkers in a biological sample of the same type obtained from thepatient prior to treatment with the complement inhibitor. In someembodiments, the patient has, is suspected of having, or is at risk fordeveloping, aHUS.

In another aspect, the disclosure features a method for determiningwhether an aHUS patient has responded to therapy with a complementinhibitor, the method comprising measuring the concentration of one ormore biomarkers of terminal complement activation set forth in Table 1or 11, e.g., C5a and/or sC5b-9, in a biological sample obtained from apatient having, suspected of having, or at risk for developing, aHUS andtreated with a complement inhibitor (e.g., an anti-C5 antibody); anddetermining that the patient has responded to the therapy if theconcentration of the one or more biomarkers in the biological sample isreduced, as compared to the concentration of the one or more biomarkersin a biological sample of the same type obtained from the patient priorto treatment with the complement inhibitor or determining that thepatient has not responded to the therapy if the concentration of the oneor more biomarkers in the biological sample is not reduced, as comparedto the concentration of the one or more biomarkers in a biologicalsample of the same type obtained from the patient prior to treatmentwith the complement inhibitor. Thus, the method can be used to assess ormonitor terminal complement blockade in an aHUS patient treated with acomplement inhibitor. In embodiments in which the patient isnon-responsive, or less responsive to therapy, the method can alsoinclude changing the dose amount or dose frequency of the complementinhibitor or electing a different complement inhibitor (e.g., aninhibitor of C3 activation) for use in treating the patient.

In another aspect, the disclosure features a method for determiningwhether an aHUS patient has responded to therapy with a complementinhibitor, the method comprising measuring the concentration of one ormore biomarkers of vascular inflammation or endothelial activation setforth in Table 1 or 11, e.g., sTNFR1, sVCAM-1, or thrombomodulin, in abiological sample obtained from a patient having, suspected of having,or at risk for developing, aHUS; and determining that the patient hasresponded to the therapy if the concentration of the one or morebiomarkers in the biological sample is reduced, as compared to theconcentration of the one or more biomarkers in a biological sample ofthe same type obtained from the patient prior to treatment with thecomplement inhibitor or determining that the patient has not respondedto the therapy if the concentration of the one or more biomarkers in thebiological sample is not reduced, as compared to the concentration ofthe one or more biomarkers in a biological sample of the same typeobtained from the patient prior to treatment with the complementinhibitor. Thus, the method can be used to assess or monitor vascularinflammation in an aHUS patient treated with a complement inhibitor. Inembodiments in which the patient is non-responsive, or less responsiveto therapy, the method can also include changing the dose amount or dosefrequency of the complement inhibitor or electing a different complementinhibitor (e.g., an inhibitor of C3 activation) for use in treating thepatient.

In another aspect, the disclosure features a method for determiningwhether an aHUS patient has responded to therapy with a complementinhibitor, the method comprising measuring the concentration of one ormore biomarkers of renal injury set forth in Table 1 or 11, e.g.,clusterin, TIMP-1, FABP-1, β2m, and/or cystatin-C, in a biologicalsample obtained from a patient having, suspected of having, or at riskfor developing, aHUS; and determining that the patient has responded tothe therapy if the concentration of the one or more biomarkers in thebiological sample is reduced, as compared to the concentration of theone or more biomarkers in a biological sample of the same type obtainedfrom the patient prior to treatment with the complement inhibitor ordetermining that the patient has not responded to the therapy if theconcentration of the one or more biomarkers in the biological sample isnot reduced, as compared to the concentration of the one or morebiomarkers in a biological sample of the same type obtained from thepatient prior to treatment with the complement inhibitor. Thus, themethod can be used to assess or monitor renal injury in an aHUS patienttreated with a complement inhibitor. In embodiments in which the patientis non-responsive, or less responsive to therapy, the method can alsoinclude changing the dose amount or dose frequency of the complementinhibitor or electing a different complement inhibitor (e.g., aninhibitor of C3 activation) for use in treating the patient.

The inventors have also discovered that, in aHUS patients, the relativeelevation of terminal complement activation markers C5a and sC5b-9concentrations (e.g., urinary concentrations) are much higher than therelative elevation of levels of complement alternative pathwayactivation markers (e.g., Ba) in these patients. That is, the medianconcentration of C5a and sC5b-9 in aHUS patients was 45 and 305 foldhigher, respectively, than the median concentration of these markers innormal healthy humans, whereas the median concentration of Ba was onlyapproximately 5-fold higher than the median concentration of Ba innormal healthy humans. While not being bound by any particular theory ormechanism of action, the inventors believe that the ratio of terminalcomplement activation over alternative pathway activation is a usefuldiagnostic tool for aHUS. Thus, in another aspect, the disclosurefeatures a method of diagnosing aHUS or confirming a diagnosis of aHUS,which method includes comparing the level of activation of terminalcomplement (e.g., sCSb-9 or C5a) to the level of activation of upstreamalternative pathway activation (e.g., Ba or Bb) (relative to normalhealthy humans), wherein a higher degree of terminal activation relativeto the alternative pathway activation is an indication that the patienthas aHUS. For example, a ratio indicative of aHUS could be, e.g.,approximately 45:5 or 305:5, fold-induction of terminal complementactivation to fold-induction alternative pathway activation. Moreover,the inventors believe that this ratio can be useful for distinguishingaHUS from other complement-associated diseases, such as, thromboticthrombocytopenic purpura (TTP), which may not exhibit such a differencein terminal complement and upstream alternative pathway activationlevels.

“Polypeptide,” “peptide,” and “protein” are used interchangeably andmean any peptide-linked chain of amino acids, regardless of length orpost-translational modification. The proteins described herein cancontain or be wild-type proteins or can be variants that have not morethan 50 (e.g., not more than one, two, three, four, five, six, seven,eight, nine, ten, 12, 15, 20, 25, 30, 35, 40, or 50) conservative aminoacid substitutions. Conservative substitutions typically includesubstitutions within the following groups: glycine and alanine; valine,isoleucine, and leucine; aspartic acid and glutamic acid; asparagine,glutamine, serine and threonine; lysine, histidine and arginine; andphenylalanine and tyrosine.

As used herein, percent (%) amino acid sequence identity is defined asthe percentage of amino acids in a candidate sequence that are identicalto the amino acids in a reference sequence, after aligning the sequencesand introducing gaps, if necessary, to achieve the maximum percentsequence identity. Alignment for purposes of determining percentsequence identity can be achieved in various ways that are within theskill in the art, for instance, using publicly available computersoftware such as BLAST software. Appropriate parameters for measuringalignment, including any algorithms needed to achieve maximal alignmentover the full-length of the sequences being compared can be determinedby known methods.

As used herein, the term “antibody” includes both whole antibodies andantigen-binding fragments of the whole antibodies. Whole antibodiesinclude different antibody isotypes including IgM, IgG, IgA, IgD, andIgE antibodies. The term “antibody” includes a polyclonal antibody, amonoclonal antibody, a chimerized or chimeric antibody, a humanizedantibody, a primatized antibody, a deimmunized antibody, and a fullyhuman antibody. The antibody can be made in or derived from any of avariety of species, e.g., mammals such as humans, non-human primates(e.g., orangutan, baboons, or chimpanzees), horses, cattle, pigs, sheep,goats, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats, andmice. The antibody can be a purified or a recombinant antibody.

As used herein, the term “antibody fragment,” “antigen-bindingfragment,” or similar terms refer to a fragment of an antibody thatretains the ability to bind to a target antigen (e.g., human C5) andinhibit the activity of the target antigen. Such fragments include,e.g., a single chain antibody, a single chain Fv fragment (scFv), an Fdfragment, an Fab fragment, an Fab′ fragment, or an F(ab′)₂ fragment. AnscFv fragment is a single polypeptide chain that includes both the heavyand light chain variable regions of the antibody from which the scFv isderived. In addition, intrabodies, minibodies, triabodies, and diabodiesare also included in the definition of antibody and are compatible foruse in the methods described herein. See, e.g., Todorovska et al. (2001)J Immunol Methods 248(1):47-66; Hudson and Kortt (1999) J ImmunolMethods 231(1):177-189; Poljak (1994) Structure 2(12):1121-1123; andRondon and Marasco (1997) Annual Review of Microbiology 51:257-283, thedisclosures of each of which are incorporated herein by reference intheir entirety. Bispecific antibodies (including DVD-Ig antibodies; seebelow) are also embraced by the term “antibody.” Bispecific antibodiesare monoclonal, preferably human or humanized, antibodies that havebinding specificities for at least two different antigens.

As used herein, the term “antibody” also includes, e.g., single domainantibodies such as camelized single domain antibodies. See, e.g.,Muyldermans et al. (2001) Trends Biochem Sci 26:230-235; Nuttall et al.(2000) Curr Pharm Biotech 1:253-263; Riechmann et al. (1999) J ImmunolMeth 231:25-38; PCT application publication nos. WO 94/04678 and WO94/25591; and U.S. Pat. No. 6,005,079, all of which are incorporatedherein by reference in their entireties. In some embodiments, thedisclosure provides single domain antibodies comprising two VH domainswith modifications such that single domain antibodies are formed.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure pertains. Preferred methods andmaterials are described below, although methods and materials similar orequivalent to those described herein can also be used in the practice ortesting of the presently disclosed methods and compositions. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.

Other features and advantages of the present disclosure, e.g., methodsfor treating complement-associated disorders in a subject, will beapparent from the following description, the examples, and from theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a dot plot depicting the concentration of C5a (in ng/mg ofurinary creatine) in the urine of aHUS patients both before treatmentwith eculizumab (Pre-Tx) and various weeks after initiating treatmentwith eculizumab. The concentration of urinary C5a was also measured inthe urine from normal, healthy individuals (NORM).

FIG. 1B is a dot plot depicting the concentration of sC5b-9 (in ng/mg ofurinary creatine) in the urine of aHUS patients both before treatmentwith eculizumab (Pre-Tx) and various weeks after initiating treatmentwith eculizumab. The concentration of urinary C5b9 was also measured inthe urine from normal, healthy individuals (NORM).

FIG. 1C is a dot plot depicting the concentration of complementcomponent Ba (in ng/mL) in the plasma of aHUS patients both beforetreatment with eculizumab (Pre-Tx) and various weeks after initiatingtreatment with eculizumab. The concentration of Ba was also measured inthe plasma from normal, healthy individuals (normals).

FIG. 1D is a bar graph depicting the mean percentage (%) reduction inurinary C5a levels (Y-axis) over time in aHUS patients (N=26) postinitiation of treatment with eculizumab. The x-axis indicates the weekof the aHUS patient visit for evaluation post-initiation of treatment,e.g., V3 is the patient visit for evaluation at week 3 post-initiationof treatment.

FIG. 1E is a bar graph depicting the mean percentage (%) reduction inurinary sC5b-9 levels (Y-axis) over time in aHUS patients (N=23) postinitiation of treatment with eculizumab. The x-axis indicates the weekof the aHUS patient visit for evaluation post-initiation of treatment,e.g., V3 is the patient visit for evaluation at week 3 post-initiationof treatment.

FIG. 1F is a bar graph depicting the mean percentage (%) reduction inplasma Ba levels (Y-axis) over time in aHUS patients (N=35) postinitiation of treatment with eculizumab. The x-axis indicates the weekof the aHUS patient visit for evaluation post-initiation of treatment,e.g., V3 is the patient visit for evaluation at week 3 post-initiationof treatment.

FIGS. 2A-2C are bar graphs depicting the percentage of aHUS patients whoachieve normalized concentrations of urinary C5a (FIG. 2A), urinarysC5b9 (FIG. 2B), and plasma Ba (FIG. 2C) at baseline (pre-treatment witheculizumab) and various weeks following initiation of treatment witheculizumab.

FIG. 3A is a dot plot depicting the concentration of prothrombinfragment 1+2 (in pmol/L) in the plasma of aHUS patients both beforetreatment with eculizumab (Pre-Tx) and various weeks after initiatingtreatment with eculizumab. The concentration of plasma F1+2 was alsomeasured in the plasma from normal, healthy individuals (normals).

FIG. 3B is a dot plot depicting the concentration of D-dimer (in μg/L)in the plasma of aHUS patients both before treatment with eculizumab(Pre-Tx) and various weeks after initiating treatment with eculizumab.The concentration of plasma D-dimer was also measured in the plasma fromnormal, healthy individuals (normals).

FIG. 3C is a bar graph depicting the mean percentage (%) reduction inplasma prothrombin fragment F1+2 levels (Y-axis) over time in aHUSpatients post initiation of treatment with eculizumab. The x-axisindicates the week of the aHUS patient visit for evaluationpost-initiation of treatment, e.g., V3 is the patient visit forevaluation at week 3 post-initiation of treatment.

FIG. 3D is a bar graph depicting the mean percentage (%) reduction inplasma d-dimer levels (Y-axis) over time in aHUS patients postinitiation of treatment with eculizumab. The x-axis indicates the weekof the aHUS patient visit for evaluation post-initiation of treatment,e.g., V3 is the patient visit for evaluation at week 3 post-initiationof treatment.

FIGS. 4A and 4B are bar graphs depicting the percentage of aHUS patientswho achieve normalized concentrations of plasma prothrombin fragment 1+2(FIG. 4A) and plasma D-dimer (FIG. 4B) at baseline (pre-treatment witheculizumab) and various weeks following initiation of treatment witheculizumab.

FIG. 5A is a dot plot depicting the concentration of thrombomodulin (inng/mL) in the plasma (EDTA treated plasma) of aHUS patients both beforetreatment with eculizumab (Pre-Tx) and various weeks after initiatingtreatment with eculizumab. The concentration of plasma thrombomodulinwas also measured in the plasma from normal, healthy individuals(normals). EOS designates the results of the analysis of samplesobtained at the “end of study”.

FIG. 5B is a dot plot depicting the concentration of VCAM-1 (in ng/mL)in the serum of aHUS patients both before treatment with eculizumab(Pre-Tx) and various weeks after initiating treatment with eculizumab.The concentration of serum VCAM-1 was also measured in the serum fromnormal, healthy individuals (normal pool). EOS designates the results ofthe analysis of samples obtained at the “end of study”.

FIG. 5C is a dot plot depicting the activity of vWF (in mU/mL) in theplasma (EDTA treated plasma) of aHUS patients both before treatment witheculizumab (Pre-Tx) and various weeks after initiating treatment witheculizumab. The activity of vWF was also measured in the plasma fromnormal, healthy individuals (normals). EOS designates the results of theanalysis of samples obtained at the “end of study”.

FIGS. 6A and 6B are bar graphs depicting the percentage of aHUS patientsthat achieve normalized plasma thrombomodulin concentrations (FIG. 6A)and plasma vWF activity levels (FIG. 4B) at baseline (pre-treatment witheculizumab) and various weeks following initiation of treatment witheculizumab.

FIG. 6C is a bar graph depicting the mean percentage (%) reduction inplasma thrombomodulin levels (Y-axis) over time in aHUS patients (N=33)post initiation of treatment with eculizumab. The x-axis indicates theweek of the aHUS patient visit for evaluation post-initiation oftreatment, e.g., V3 is the patient visit for evaluation at week 3post-initiation of treatment.

FIG. 6D is a bar graph depicting the mean percentage (%) reduction inserum VCAM-1 levels (Y-axis) over time in aHUS patients (N=36) postinitiation of treatment with eculizumab. The x-axis indicates the weekof the aHUS patient visit for evaluation post-initiation of treatment,e.g., V3 is the patient visit for evaluation at week 3 post-initiationof treatment.

FIG. 7A is a dot plot depicting the concentration of TNFR1 (in pg/mL) inthe serum of aHUS patients both before treatment with eculizumab(Pre-Tx) and various weeks after initiating treatment with eculizumab.The concentration of serum TNFR1 was also measured in the serum fromnormal, healthy individuals (normal pool). EOS designates the results ofthe analysis of samples obtained at the “end of study”.

FIG. 7B is a bar graph depicting the percentage of aHUS patients thatachieve normalized serum TNFR1 concentrations at baseline (pre-treatmentwith eculizumab) and various weeks following initiation of treatmentwith eculizumab.

FIG. 8A is a dot plot depicting the concentration of cystatin C (CysC)(in ng/mg of urinary creatine) in the urine of aHUS patients both beforetreatment with eculizumab (Pre-Tx) and various weeks after initiatingtreatment with eculizumab. The concentration of urinary CysC was alsomeasured in the urine from normal, healthy individuals (NORM).

FIG. 8B is a dot plot depicting the concentration of β2M (in μg/mg ofurinary creatine) in the urine of aHUS patients both before treatmentwith eculizumab (Pre-Tx) and various weeks after initiating treatmentwith eculizumab. The concentration of urinary β2M was also measured inthe urine from normal, healthy individuals (NORM).

FIG. 8C is a dot plot depicting the concentration of NGAL (in ng/mg ofurinary creatine) in the urine of aHUS patients both before treatmentwith eculizumab (Pre-Tx) and various weeks after initiating treatmentwith eculizumab. The concentration of urinary NGAL was also measured inthe urine from normal, healthy individuals (NORM).

FIGS. 9A-9E are a series of bar graphs depicting the mean levels ofseveral aHUS biomarker proteins in aHUS patients that were subjected todialysis (Dialysis), as compared to those aHUS patients that were notsubjected to dialysis (no Dialysis) prior to enrollment in the studydescribed herein. FIG. 9A depicts the mean concentration of serum TNFR1(in pg/mL); FIG. 9B depicts the mean concentration of urinary β2M (inμg/mg of urinary creatine); FIG. 9C depicts the concentration of plasmaBa (in ng/mL); FIG. 9D depicts the concentration of urinary sC5b9 (inng/mg of urinary creatine); and FIG. 9E depicts the concentration ofurinary C5a (in ng/mL).

FIG. 10A is a dot plot depicting the concentration of serum TNFR1 (inpg/mL) in aHUS patients exhibiting stable clinical parameters (clinicalremission) (Without TMA) and those aHUS patients that continue toexperience elevated haptoglobin and LDH levels (and reduced plateletcounts) (Others), both at baseline and at 1 to 2.5 weeks post initiationof treatment with eculizumab. Also shown are the concentrations of serumTNFR1 from normal, healthy individuals (Normals).

FIGS. 10B-10E are a series of bar graphs, each one depicting theconcentration of a given biomarker in patients with normal hematologicmarkers LDH and haptoglobin (“normal patients” or patients deemed to bein clinical remission), patients with abnormal (elevated) hematologicmarkers (“abnormal patients” or patients with active aHUS presentation),and healthy subjects (“normals”). FIG. 10B depicts the levels of plasmaBa (ng/mL) in these subject populations. FIG. 10C depicts the level ofserum VCAM-1 (ng/mL) in the subject populations. FIG. 10E depicts thelevel of plasma prothrombin fragments 1+2 (pmol/L) in the populations,and FIG. 10D depicts the level of plasma D-dimer (in μg/L). The P valuesfor the respective group comparisons are shown in the figures.

FIGS. 10E-10I are a series of bar graphs, each one depicting theconcentration of a given biomarker in patients with normal plateletlevels (“normal patients”), patients with abnormal (reduced) plateletlevels (“abnormal patients”), and healthy subjects (“normals”). FIG. 10Fdepicts the levels of plasma Ba (ng/mL) in these subject populations.FIG. 10G depicts the level of serum VCAM-1 (ng/mL) in the subjectpopulations. FIG. 10I depicts the level of plasma prothrombin fragments1+2 (pmol/L) in the populations, and FIG. 10H depicts the level ofplasma D-dimer (in μg/L). The P values for the respective groupcomparisons are shown in the figures.

FIG. 11 is a bar graph depicting the mean percentage change in serumTNFR1 and urinary clusterin, C5a, and C5b9 levels in those aHUS patientswho achieve a complete TMA response and those patients who stillexperience TMA events (incomplete response). (As noted and elaborated onin the working examples, a complete TMA response refers to anormalization of hematologic parameters and preservation of renalfunction.)

FIG. 12 is a bar graph depicting the mean percentage change in plasma Balevels in eculizumab-treated aHUS patients experiencing a complete TMAresponse and those eculizumab-treated aHUS patients who do not (others).

FIG. 13 is bar graph depicting the mean change from baseline (initialvisit, prior to treatment with eculizumab) in platelet count (10⁹/L) atweeks 12-17 and week 26 post initiation of treatment with eculizumab inaHUS patients with normalized levels of plasma Ba versus persistentlyelevated plasma Ba levels. The p values for each observation are alsoprovided in the figure.

FIGS. 14A-14D are a series of bar graphs depicting the observation thatcertain aHUS-associated biomarkers are elevated in aHUS patients withabnormal TMA markers at baseline. FIG. 14A depicts the concentration ofcystatin C (CysC) (in ng/mg of urinary creatine) in the urine of aHUSpatients with normal platelet counts (>150,000 per μL of blood) ascompared to patients with reduced platelet counts (<150,000 per μL ofblood). FIG. 14B depicts the concentration of clusterin (in ng/mg ofurinary creatine) in the urine of aHUS patients with normal plateletcounts (>150,000 per μL of blood) as compared to patients with reducedplatelet counts (<150,000 per μL of blood). FIG. 14C depicts theconcentration of VCAM-1 in the serum of aHUS patients with normal LDHlevels as compared to patients with elevated LDH levels. FIG. 14Ddepicts the concentration of d-dimer (in μg/L) in the plasma of aHUSpatients with normal LDH levels as compared to patients with elevatedLDH levels. The p values for each observation are indicated in thefigures.

FIG. 15 is a bar graph depicting the level of cystatin C (ng/mg ofurinary creatine) in the urine of aHUS patients at baseline havingrepeated plasma therapy (Repeated PT; N=23), no plasma therapy (No PT;N=3), or in normal patients (N=9).

FIG. 16 is a series of bar graphs depicting the mean change in baselineeGFR (mL/min/1.73 m²) in aHUS patients who achieve normalized levels ofvarious biomarkers (plasma Ba, serum VCAM-1, plasma F1+2, plasmad-dimer, and urinary cystatin C) following eculizumab treatment ascompared to aHUS patients in whom the concentration of these biomarkersremain elevated.

FIGS. 17A-E are a series of bar graphs depicting the observation thatcertain aHUS-associated biomarkers are elevated in aHUS patients priorto treatment with a complement inhibitor, regardless of whether thepatients have received plasma exchange (PE) or plasma infusion (PI)therapy. FIG. 17A depicts the concentration of Factor B proteolyticfragment Ba (in ng/mL) in the plasma of normal healthy volunteers (NHV),aHUS patients receiving PE or PI therapy (PE/PI), or aHUS patients notreceiving PE/PI therapy (no PE/PI). FIG. 17B depicts the concentrationof sTNFR1 (in pg/mL) in the serum of normal healthy volunteers (NHV),aHUS patients receiving PE or PI therapy (PE/PI), or aHUS patients notreceiving PE/PI therapy (no PE/PI). FIG. 17C depicts the concentrationof sVCAM-1 (in ng/mL) in the serum of normal healthy volunteers (NHV),aHUS patients receiving PE or PI therapy (PE/PI), or aHUS patients notreceiving PE/PI therapy (no PE/PI). FIG. 17D depicts the concentrationof D-dimer (in μg/L) in the plasma of normal healthy volunteers (NHV),aHUS patients receiving PE or PI therapy (PE/PI), or aHUS patients notreceiving PE/PI therapy (no PE/PI). FIG. 17E depicts the concentrationof cystatin-C (in ng/mg of urinary creatinine) in the urine of normalhealthy volunteers (NHV), aHUS patients receiving PE or PI therapy(PE/PI), or aHUS patients not receiving PE/PI therapy (no PE/PI). The pvalues for each observation are indicated in the figures.

FIGS. 18A-E are a series of bar graphs depicting the observation thatcertain aHUS-associated biomarkers are elevated in aHUS patients priorto treatment with a complement inhibitor, regardless of platelet levelsin the patients. FIG. 18A depicts the concentration of Factor Bproteolytic fragment Ba (in ng/mL) in the plasma of normal healthyvolunteers (NHV), aHUS patients having normal platelet levels(>150×10⁹), or aHUS patients having reduced platelet counts (<150×10⁹).FIG. 18B depicts the concentration of sTNFR1 (in pg/mL) in the serum ofnormal healthy volunteers (NHV), aHUS patients having normal plateletlevels (>150×10⁹), or aHUS patients having reduced platelet counts(<150×10⁹). FIG. 18C depicts the concentration of sVCAM-1 (in ng/mL) inthe serum of normal healthy volunteers (NHV), aHUS patients havingnormal platelet levels (>150×10⁹), or aHUS patients having reducedplatelet counts (<150×10⁹). FIG. 18D depicts the concentration ofD-dimer (in μg/L) in the plasma of normal healthy volunteers (NHV), aHUSpatients having normal platelet levels (>150×10⁹), or aHUS patientshaving reduced platelet counts (<150×10⁹). FIG. 18E depicts theconcentration of cystatin-C (in ng/mg of urinary creatinine) in theurine of normal healthy volunteers (NHV), aHUS patients having normalplatelet levels (>150×10⁹), or aHUS patients having reduced plateletcounts (<150×10⁹). The p values for each observation are indicated inthe figures.

FIGS. 19A-E are a series of bar graphs depicting the observation thatcertain aHUS-associated biomarkers are elevated in aHUS patients priorto treatment with a complement inhibitor, regardless of haptoglobin (Hp)or lactate dehydrogenase (LDH) levels. FIG. 19A depicts theconcentration of Factor B proteolytic fragment Ba (in ng/mL) in theplasma of normal healthy volunteers (NHV), aHUS patients having normalHp and LDH levels, or aHUS patients having elevated (abnormal) Hp/LDH.FIG. 19B depicts the concentration of sTNFR1 (in pg/mL) in the serum ofnormal healthy volunteers (NHV), aHUS patients having normal Hp and LDHlevels, or aHUS patients having elevated (abnormal) Hp/LDH. FIG. 19Cdepicts the concentration of sVCAM-1 (in ng/mL) in the serum of normalhealthy volunteers (NHV), aHUS patients having normal Hp and LDH levels,or aHUS patients having elevated (abnormal) Hp/LDH. FIG. 19D depicts theconcentration of D-dimer (in μg/L) in the plasma of normal healthyvolunteers (NHV), aHUS patients having normal Hp and LDH levels, or aHUSpatients having elevated (abnormal) Hp/LDH. FIG. 19E depicts theconcentration of cystatin-C (in ng/mg of urinary creatinine) in theurine of normal healthy volunteers (NHV), aHUS patients having normal Hpand LDH levels, or aHUS patients having elevated (abnormal) Hp/LDH. Thep values for each observation are indicated in the figures.

FIGS. 20A-B are Box-Whisker plots depicting the longitudinal effects ofsustained eculizumab treatment on terminal complement activation in aHUSpatients. FIG. 20A depicts the change over time in the concentration ofurinary C5a (ng/mg of urinary creatinine) of aHUS patients followingeculizumab treatment, as compared to the concentration of urinary C5a inthe urine of normal healthy volunteers (NHV). FIG. 20B depicts thechange over time in the concentration of urinary sC5b-9 (ng/mg ofurinary creatinine) of aHUS patients following eculizumab treatment, ascompared to the concentration of urinary sC5b-9 in the urine of normalhealthy volunteers (NHV). The Box-Whisker plots show median, 25^(Th),and 75^(th) percentiles and range. *First time point at which levelswere significantly reduced vs. baseline (BL); P values versus baselineat each timepoint were calculated using a restricted maximumlikelihood-based repeated measures approach (Mixed Model). P valuescompared with NHV were calculated using the Wilcoxon Rank Sum test.

FIGS. 21A-C are Box-Whisker plots depicting the longitudinal effects ofsustained eculizumab treatment on the concentration of biomarkerproteins associated with renal injury in aHUS patients. FIG. 20A depictsthe change over time in the concentration of urinary FABP-1 (ng/mg ofurinary creatinine) of aHUS patients following eculizumab treatment, ascompared to the concentration of urinary FABP-1 in the urine of normalhealthy volunteers (NHV). FIG. 21B depicts the change over time in theconcentration of urinary cystatin-C (ng/mg of urinary creatinine) ofaHUS patients following eculizumab treatment, as compared to theconcentration of urinary cystatin-C in the urine of normal healthyvolunteers (NHV). FIG. 21C depicts the change over time in theconcentration of urinary clusterin (ng/mg of urinary creatinine) of aHUSpatients following eculizumab treatment, as compared to theconcentration of urinary clusterin in the urine of normal healthyvolunteers (NHV). The Box-Whisker plots show median, 25^(Th), and75^(th) percentiles and range. *First time point at which levels weresignificantly reduced vs. baseline (BL); P values versus baseline ateach timepoint were calculated using a restricted maximumlikelihood-based repeated measures approach (Mixed Model). P valuescompared with NHV were calculated using the Wilcoxon Rank Sum test.

FIG. 22 is a Box-Whisker plot depicting the longitudinal effects ofsustained eculizumab treatment on complement alternative pathwayactivation in aHUS patients. The change over time in the concentrationof Ba (ng/mL) in the plasma of aHUS patients following eculizumabtreatment is shown along with the concentration of plasma Ba in normalhealthy volunteers (NHV). The Box-Whisker plot shows median, 25^(th),and 75^(th) percentiles and range. *First time point at which levelswere significantly reduced vs. baseline (BL); P values versus baselineat each timepoint were calculated using a restricted maximumlikelihood-based repeated measures approach (Mixed Model). P valuescompared with NHV were calculated using the Wilcoxon Rank Sum test.

FIGS. 23A-C are Box-Whisker plots depicting the longitudinal effects ofsustained eculizumab treatment on the concentration of biomarkerproteins associated with inflammation, endothelial cell activation, andtissue damage in aHUS patients. FIG. 23A depicts the change over time inthe concentration of sTNFR1 (pg/mL) in the serum of aHUS patientsfollowing eculizumab treatment, as compared to the concentration ofsTNFR1 in the serum of normal healthy volunteers (NHV). FIG. 23B depictsthe change over time in the concentration of sVCAM-1 (ng/mL) in theserum of aHUS patients following eculizumab treatment, as compared tothe concentration of the analyte in the serum of normal healthyvolunteers (NHV). FIG. 23C depicts the change over time in theconcentration of thrombomodulin (ng/mL) in the plasma of aHUS patientsfollowing eculizumab treatment, as compared to the concentration of theanalyte in the plasma of normal healthy volunteers (NHV). TheBox-Whisker plots show median, 25^(Th), and 75^(th) percentiles andrange. *First time point at which levels were significantly reduced vs.baseline (BL); P values versus baseline at each timepoint werecalculated using a restricted maximum likelihood-based repeated measuresapproach (Mixed Model). P values compared with NHV were calculated usingthe Wilcoxon Rank Sum test.

FIGS. 24A-B are Box-Whisker plots depicting the longitudinal effects ofsustained eculizumab treatment on the concentration of biomarkerproteins associated with thrombosis and coagulation in aHUS patients.FIG. 24A depicts the change over time in the concentration of F1+2(pmol/L) in the plasma of aHUS patients following eculizumab treatment,as compared to the concentration of the analyte in the plasma of normalhealthy volunteers (NHV). FIG. 24B depicts the change over time in theconcentration of D-dimer (μg/L) in the plasma of aHUS patients followingeculizumab treatment, as compared to the concentration of the analyte inthe plasma of normal healthy volunteers (NHV). The Box-Whisker plotsshow median, 25^(Th), and 75^(th) percentiles and range. *First timepoint at which levels were significantly reduced vs. baseline (BL); Pvalues versus baseline at each timepoint were calculated using arestricted maximum likelihood-based repeated measures approach (MixedModel). P values compared with NHV were calculated using the WilcoxonRank Sum test.

OVERVIEW OF THE COMPLEMENT SYSTEM

The complement system acts in conjunction with other immunologicalsystems of the body to defend against intrusion of cellular and viralpathogens. There are at least 25 complement proteins, which are found asa complex collection of plasma proteins and membrane cofactors. Theplasma proteins make up about 10% of the globulins in vertebrate serum.Complement components achieve their immune defensive functions byinteracting in a series of intricate but precise enzymatic cleavage andmembrane binding events. The resulting complement cascade leads to theproduction of products with opsonic, immunoregulatory, and lyticfunctions. A concise summary of the biologic activities associated withcomplement activation is provided, for example, in The Merck Manual,16^(th) Edition.

The complement cascade progresses via the classical pathway, thealternative pathway, or the lectin pathway. These pathways share manycomponents, and while they differ in their initial steps, they convergeand share the same “terminal complement” components (C5 through C9)responsible for the activation and destruction of target cells.

The classical pathway (CP) is typically initiated by antibodyrecognition of, and binding to, an antigenic site on a target cell. Thealternative pathway (AP) can be antibody independent, and can beinitiated by certain molecules on pathogen surfaces. Additionally, thelectin pathway is typically initiated with binding of mannose-bindinglectin (MBL) to high mannose substrates. These pathways converge at thepoint where complement component C3 is cleaved by an active protease toyield C3a and C3b. Other pathways activating complement attack can actlater in the sequence of events leading to various aspects of complementfunction. C3a is an anaphylatoxin. C3b binds to bacterial and othercells, as well as to certain viruses and immune complexes, and tags themfor removal from the circulation. This opsonic function of C3b isgenerally considered to be the most important anti-infective action ofthe complement system. C3b also forms a complex with other componentsunique to each pathway to form classical or alternative C5 convertase,which cleaves complement component C5 (hereinafter referred to as “C5”)into C5a and C5b.

Cleavage of C5 releases biologically active species such as for exampleC5a, a potent anaphylatoxin and chemotactic factor, and C5b whichthrough a series of protein interactions leads to the formation of thelytic terminal complement complex, C5b-9. C5a and C5b-9 also havepleiotropic cell activating properties, by amplifying the release ofdownstream inflammatory factors, such as hydrolytic enzymes, reactiveoxygen species, arachidonic acid metabolites and various cytokines.

C5b combines with C6, C7, and C8 to form the C5b-8 complex at thesurface of the target cell. Upon binding of several C9 molecules, themembrane attack complex (MAC, C5b-9, terminal complement complex—TCC) isformed. When sufficient numbers of MACs insert into target cellmembranes the openings they create (MAC pores) mediate rapid osmoticlysis of the target cells. Lower, non-lytic concentrations of MACs canproduce other effects. In particular, membrane insertion of smallnumbers of the C5b-9 complexes into endothelial cells and platelets cancause deleterious cell activation. In some cases activation may precedecell lysis.

As mentioned above, C3a and C5a are activated complement components.These can trigger mast cell degranulation, which releases histamine frombasophils and mast cells, and other mediators of inflammation, resultingin smooth muscle contraction, increased vascular permeability, leukocyteactivation, and other inflammatory phenomena including cellularproliferation resulting in hypercellularity. C5a also functions as achemotactic peptide that serves to attract pro-inflammatory granulocytesto the site of complement activation. C5a receptors are found on thesurfaces of bronchial and alveolar epithelial cells and bronchial smoothmuscle cells. C5a receptors have also been found on eosinophils, mastcells, monocytes, neutrophils, and activated lymphocytes.

DETAILED DESCRIPTION

As described herein and exemplified in the working Examples, theinventors identified biomarkers for aHUS. For example, it has beendiscovered that an elevated or, in some cases, reduced concentration ofcertain proteins is associated with the presence of aHUS. Similarly, areduced or elevated concentration (or activity) of certain proteins in abiological fluid obtained from an aHUS patient treated with a complementinhibitor indicates that the patient has responded to therapy with theinhibitor. Accordingly, analysis of the concentration and/or activitylevel of such proteins can be employed to evaluate, among other things,risk for aHUS, diagnose aHUS, monitor progression or abatement of aHUS,and/or monitor treatment response to a complement inhibitor.

aHUS Biomarker Proteins and Applications

aHUS biomarker proteins (as well as exemplary biological fluids in whichthey are found) are set forth in Table 1. The protein sequenceassociated with the name of each of the biomarkers listed in Table 1 inGenBank (National Center for Biotechnology Information (NCBI)) asavailable as of the filing date of the present application areincorporated herein by reference.

TABLE 1 Tissue Source NCBI Reference Biomarker Abbr. Serum Plasma UrineSeq no.* Markers of Inflammation/platelet or endothelial activationChemokine (C-X- CXCL9 X NP_002407.1 C motif) ligand 9 Chemokine (C-X-CXCL-10 X NP_001556.2 C motif) ligand 10 Interleukin-1 beta IL-1β XNP_000567.1 Interleukin-6 IL-6 X NP_000591.1 Interleukin-8 IL-8 XNP_000575.1 Interleukin-12 p70 IL-12p70 X NP_000873.2 (p35) NP_002178.2(p40) Interferon-gamma IFN-γ X NP_000610.2 platelet-selectin p-selectinX NP_002996.2 endothelial- e-selectin X NP_000441.2 selectinIntercellular ICAM-1 X NP_000192.2 Adhesion Molecule-1 Vascular cellVCAM-1 X NP_001069.1 adhesion molecule-1 Monocyte MCP-1 X NP_002973.1chemotactic protein-1 Vascular VEGF X NP_001020537.2 endothelial growthfactor Regulated on CCL5 X NP_002976.2 Activation, Normal T cellExpressed and Secreted (CCL5) Soluble CD40 sCD40L X NP_000065.1** ligandSoluble Tumor sTNFR1 X NP_001056.1** necrosis factor receptor 1Interleukin-18 IL-18 X NP_001553.1 Markers of Inflammation/Renal Injuryneutrophil NGAL X NP_005555.2 gelatinase- associated lipocalin Kidneyinjury KIM-1 X NP_001092884.1 molecule-1 Osteopontin OPN XNP_001035147.1 tissue inhibitor TIMP-1 X NP_003245.1 of metallo-proteinases- 1 Interleukin-18 IL-18 X Supra Chemokine (C-X- CXCL9 XSupra C motif) ligand 9 Chemokine (C-X- CXCL10 X Supra C motif) ligand10 clusterin CLU X NP_001822.3 Cystatin C CyC X NP_000090.1 albumin ALBX NP_000468.1 Liver-fatty acid L-FABP X NP_001434.1 binding proteinBeta-2- β2M X NP_004039.1 microglobulin Trefoil factor 3 TFF-3 XNP_003217.3 N-acetyl-beta-D- NAG X NP_000511.2 glucosaminidaseπ-glutathione S- π-GST X NP_000843.1 transferase Alpha-glutathione α-GSTX NP_665683.1 S-transferase Complement Complement Ba Ba X SEQ ID NO: 1;See also FIG. 2 of Morley and Campbel l(1984) EMBO J 3(1):153-157.Complement C3a C3a X SEQ ID NO: 2 Complement C5a C5a X X SEQ ID NO: 3Soluble MAC sC5b9 X X NA CH₅₀ (hemolysis) CH₅₀ X NA Complement C5 C5 X XNP_001726.2 Thrombosis/coagulation D-dimer D-dimer X P02671***Prothrombin F1 + 2 X Activation F1 + 2 fragment 1 (SEQ ID NO: 4)corresponds to amino acids 44- 198 of SEQ ID NO: 6. Activation fragment2 (SEQ ID NO: 5) corresponds to amino acids 199- 327 of SEQ ID NO: 4.Von Willebrand vWF X NP_000543.2 factor Von Willebrand vWF X Id. factoractivity activity Thrombomodulin TM X NP_000352.1 *The NCBI accessionnumber for an exemplary human sequence is provided for each biomarkerprotein recited in the Table. **The soluble form of the receptor isgenerated by proteolytic processing of the membrane bound form of thereceptor. ***UniProtKB (consortium: European Bioinformatics Institute,Cambridge, UK; Swiss Institute of Bioinformatics; Geneva, Switzerland;and Protein Information Resource, Washington, D.C.) designation forhuman fibrinogen alpha, which is cleaved by thrombin to form fibrin.D-dimer is a degradation product of fibrin. A description of thecleavage-based transition of fibrinogen to fibrin to D-dimer is setforth in Soheir et al. (2009) Blood 113(13):2878-2887, the disclosure ofwhich at least as it relates to the formation of D-dimer is incorporatedherein in its entirety.

Biomarkers provided herein can be used alone or in combination as anindicator to, e.g., evaluate risk for developing aHUS, diagnosing aHUS,determining whether a subject is experiencing the first acutepresentation of aHUS, monitoring progression or abatement of aHUS,and/or monitoring response to treatment with a complement inhibitor oroptimizing such treatment. In some embodiments, an individual aHUSbiomarker protein described herein may be used. In some embodiments, atleast two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more) aHUSbiomarker proteins selected from Table 1 may be used in combination as apanel.

In some embodiments, the aHUS biomarker proteins are selected from aproteolytic fragment of complement component factor B (e.g., Ba or Bb),soluble C5b9 (sC5b9), thrombomodulin, VCAM-1, von Willebrand Factor(vWF), soluble CD40 ligand (sCD40L), prothrombin fragment F1+2, D-dimer,CXCL10, MCP-1, TNFR1, IFN-γ, ICAM-1, IL-1 beta, IL-12 p70, complementcomponent C5a, β2 microglobulin (β2M), clusterin, cystatin C, NAG,TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), CXCL9, KIM-1,IL-18, vascular endothelial cell growth factor (VEGF), IL-6, albumin,IL-8, and CCL5. The concentration and/or activity of one or more of thebiomarkers in Table 1 (or any of the subsets of biomarkers mentionedherein) can be measured.

In some embodiments, an elevation in the d-dimer concentration, relativeto the concentration of d-dimer in a normal control sample, and anelevation in the FABP-1 concentration, relative to the concentration ofFABP-1 in a normal control sample, indicates that the aHUS patient isexperiencing a first acute aHUS manifestation. In some instances, thatelevation of one or both of these aHUS biomarker proteins is asignificant elevation as compared to the normal control.

In some embodiments, an elevation in the concentration of one or more ofTNFR1, Ba, C5b-9, F1+2, β2M, clusterin, TIMP-1, NGAL, CysC, and C5a (seeTable 7) in a biological sample obtained from an aHUS patient, relativeto the control concentration of the analytes obtained, e.g., from a poolof samples from aHUS patients who have not received repeated dialysis,indicates that the patient is one who has received repeated dialysis.

In some embodiments, an elevation in the concentration of one or both ofC5a and FABP-1 (e.g., urinary C5a and FABP-1) in a biological sampleobtained from an aHUS patient, relative to the control concentration ofthe analytes obtained, e.g., from a pool of samples from aHUS patientswho have not received a kidney transplant, indicates that the patient isone who has received a kidney transplant.

In some embodiments, an elevation in the concentration of cystatin C(e.g., urinary cystatin C) in a biological sample obtained from an aHUSpatient, relative to the control concentration of the analytes obtained,e.g., from a pool of samples from aHUS patients who have not receivedrepeated plasma therapy, indicates that the patient is one who hasreceived repeated plasma therapy.

In some embodiments, a post-treatment reduction in Ba concentration(e.g., plasma Ba concentration) of at least 10 (e.g., at least 15, 20,25, 30, 35, 40, 45, or 50) %, relative to the Ba concentration in asample of the same type of biological fluid obtained from the subjectprior to treatment, indicates that the subject has or is likely toachieve a complete thrombomicroangiopathy (TMA) response (i.e.,cessation of TMA events). In some embodiments, the reduction occurs byweek 12 following the first treatment with the complement inhibitor. Insome embodiments, the reduction occurs within weeks 12-17 following thefirst treatment with the complement inhibitor. In some embodiments, thereduction occurs by week 26 following the first treatment with thecomplement inhibitor.

In some embodiments, a post-treatment reduction in one or both of CCL5and sCD40L of at least 10 (e.g., at least 15, 20, 25, 30, 35, 40, 45, or50) %, relative to the respective concentration in sample(s) of the sametype of biological fluid obtained from the subject prior to treatment,indicates that the subject has or is likely to achieve increasedplatelet counts (e.g., platelet recovery). In some embodiments, apost-treatment reduction in Ba concentration (e.g., plasma Baconcentration) of at least 10 (e.g., at least 15, 20, 25, 30, 35, 40,45, or 50) % (or normalization of Ba concentrations), relative to the Baconcentration in a sample of the same type of biological fluid obtainedfrom the subject prior to treatment, indicates that the subject has oris likely to have achieved a complete thrombomicroangiopathy (TMA)response (i.e., cessation of TMA events).

In some embodiments, the status of one or more of the aHUS biomarkersdescribed herein can be predictive of improvement in the estimatedglomerular filtration rate (eGFR) for an aHUS patient treated with acomplement inhibitor. For example, a reduction in the concentration ofprothrombin F1+2 (e.g., within 4, 5, or 6 weeks post initial treatmentin a chronic treatment regimen) and/or d-dimer (e.g., within 12, 13, 14,15, 16, or 17 weeks post initial treatment in a chronic treatmentregimen) indicates that an aHUS patient treated with a complementinhibitor has achieved or is likely to achieve a clinically meaningfulimprovement in eGFR. Achievement or likely achievement of a clinicallymeaningful improvement in eGFR is also indicated by a normalization ofIL-6 and IFN-γ concentration (e.g., within 4, 5, or 6 weeks post initialtreatment with a complement inhibitor in a chronic treatment regimen).Achievement or likely achievement of a clinically meaningful improvementin eGFR is also indicated by a normalization of Ba, CXCL9, CXCL10, andvWF concentration (e.g., within 12, 13, 14, 15, 16, or 17 weeks postinitial treatment with a complement inhibitor in a chronic treatmentregimen). In some embodiments, achievement or likely achievement of aclinically meaningful improvement in eGFR is also indicated by anormalization of Ba, CXCL9, CXCL10, β2M (e.g., in urine), CysC (e.g., inurine), vWF, d-dimer, clusterin (e.g., in urine), and/or FABP-1 (e.g.,in urine) concentration (e.g., within 26 weeks post initial treatmentwith a complement inhibitor in a chronic treatment regimen).

Methods for monitoring or evaluating the status of one or more atypicalhemolytic uremic syndrome (aHUS)-associated biomarker proteins in asubject (e.g., a mammal, e.g., a human) include: measuring in abiological fluid obtained from the subject one or both of (i) theconcentration of at least one (e.g., at least two, three, four, five,six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20)aHUS-associated biomarker protein in the biological fluid.

Measuring or determining protein expression levels in a biologicalsample may be performed by any suitable method (see, e.g., Harlow andLane (1988) “Antibodies: A Laboratory Manual”, Cold Spring HarborLaboratory: Cold Spring Harbor, N.Y.). In general, protein levels aredetermined by contacting a biological sample obtained from a subjectwith binding agents for one or more of the aHUS biomarker proteins;detecting, in the sample (e.g., the biological fluid), the levels of oneor more of the aHUS biomarker proteins that bind to the binding agents;and comparing the levels of one or more of the aHUS biomarker proteinsin the sample with the levels of the corresponding protein biomarkers ina control sample (e.g., a normal sample). In certain embodiments, asuitable binding agent is a ribosome, with or without a peptidecomponent, an RNA molecule, or a polypeptide (e.g., a polypeptide thatcomprises a polypeptide sequence of a protein marker, a peptide variantthereof, or a non-peptide mimetic of such a sequence).

Suitable binding agents also include an antibody specific for an aHUSbiomarker protein described herein (e.g., an antibody specific for anybiomarker listed in Table 1). Suitable antibodies for use in the methodsof the present invention include monoclonal and polyclonal antibodiesand antigen-binding fragments (e.g., Fab fragments or scFvs) ofantibodies. Antibodies, including monoclonal and polyclonal antibodies,fragments and chimeras, may be prepared using methods known in the art(see, for example, Kohler and Milstein (1975) Nature 256:495-497; Kozboret al. (1985) J Immunol Methods 81:31-42; Cote et al. (1983) Proc NatlAcad Sci USA 80:2026-203; and Zhang et al. (2002) J Biol Chem277:39379-39387). Antibodies to be used in the methods of the inventioncan be purified by methods well known in the art. Antibodies may also beobtained from commercial sources.

In certain embodiments, the binding agent is directly or indirectlylabeled with a detectable moiety. The role of a detectable agent is tofacilitate the detection step of the diagnostic method by allowingvisualization of the complex formed by binding of the binding agent tothe protein marker (or fragment thereof). The detectable agent can beselected such that it generates a signal that can be measured and whoseintensity is related (preferably proportional) to the amount of proteinmarker present in the sample being analyzed. Methods for labelingbiological molecules such as polypeptides and antibodies are well-knownin the art. Any of a wide variety of detectable agents can be used inthe practice of the present invention. Suitable detectable agentsinclude, but are not limited to: various ligands, radionuclides,fluorescent dyes, chemiluminescent agents, microparticles (such as, forexample, quantum dots, nanocrystals, phosphors and the like), enzymes(such as, e.g., those used in an ELISA, i.e., horseradish peroxidase,beta-galactosidase, luciferase, alkaline phosphatase), colorimetriclabels, magnetic labels, and biotin, digoxigenin or other haptens andproteins for which antisera or monoclonal antibodies are available.

In certain embodiments, the binding agents (e.g., antibodies) may beimmobilized on a carrier or support (e.g., a bead, a magnetic particle,a latex particle, a microtiter plate well, a cuvette, or other reactionvessel). Examples of suitable carrier or support materials includeagarose, cellulose, nitrocellulose, dextran, Sephadex®, Sepharose®,liposomes, carboxymethyl cellulose, polyacrylamides, polystyrene,gabbros, filter paper, magnetite, ion-exchange resin, plastic film,plastic tube, glass, polyamine-methyl vinyl-ether-maleic acid copolymer,amino acid copolymer, ethylene-maleic acid copolymer, nylon, silk, andthe like. Binding agents may be indirectly immobilized using secondbinding agents specific for the first binding agents (e.g., mouseantibodies specific for the protein markers may be immobilized usingsheep anti-mouse IgG Fc fragment specific antibody coated on the carrieror support).

Protein expression levels in a biological sample may be determined usingimmunoassays. Examples of such assays are time resolved fluorescenceimmunoassays (TR-FIA), radioimmunoas says, enzyme immunoassays (e.g.,ELISA), immunofluorescence immunoprecipitation, latex agglutination,hemagglutination, Western blot, and histochemical tests, which areconventional methods well-known in the art. Methods of detection andquantification of the signal generated by the complex formed by bindingof the binding agent with the protein marker will depend on the natureof the assay and of the detectable moiety (e.g., fluorescent moiety).

In one example, the presence or amount of protein expression of a gene(e.g., an aHUS biomarker protein depicted in Table 1) can be determinedusing a Western blotting technique. For example, a lysate can beprepared from a biological sample, or the biological sample (e.g.,biological fluid) itself, can be contacted with Laemmli buffer andsubjected to sodium-dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE). SDS-PAGE-resolved proteins, separated by size, can then betransferred to a filter membrane (e.g., nitrocellulose) and subjected toimmunoblotting techniques using a detectably-labeled antibody specificto the protein of interest. The presence or amount of bounddetectably-labeled antibody indicates the presence or amount of proteinin the biological sample.

In another example, an immunoassay can be used for detecting and/ormeasuring the protein expression of an aHUS biomarker protein (e.g., onedepicted in Table 1). As above, for the purposes of detection, animmunoassay can be performed with an antibody that bears a detectionmoiety (e.g., a fluorescent agent or enzyme). Proteins from a biologicalsample can be conjugated directly to a solid-phase matrix (e.g., amulti-well assay plate, nitrocellulose, agarose, Sepharose®, encodedparticles, or magnetic beads) or it can be conjugated to a first memberof a specific binding pair (e.g., biotin or streptavidin) that attachesto a solid-phase matrix upon binding to a second member of the specificbinding pair (e.g., streptavidin or biotin). Such attachment to asolid-phase matrix allows the proteins to be purified away from otherinterfering or irrelevant components of the biological sample prior tocontact with the detection antibody and also allows for subsequentwashing of unbound antibody. Here, as above, the presence or amount ofbound detectably-labeled antibody indicates the presence or amount ofprotein in the biological sample.

Alternatively, the protein expression levels may be determined usingmass spectrometry based methods or image-based methods known in the artfor the detection of proteins. Other suitable methods include 2D-gelelectrophoresis, proteomics-based methods such as the identification ofindividual proteins recovered from the gel (e.g., by mass spectrometryor N-terminal sequencing) and/or bioinformatics.

Methods for detecting or measuring protein expression can, optionally,be performed in formats that allow for rapid preparation, processing,and analysis of multiple samples. This can be, for example, inmulti-well assay plates (e.g., 96 wells or 386 wells) or arrays (e.g.,protein chips). Stock solutions for various reagents can be providedmanually or robotically, and subsequent sample preparation, pipetting,diluting, mixing, distribution, washing, incubating (e.g.,hybridization), sample readout, data collection (optical data) and/oranalysis (computer aided image analysis) can be done robotically usingcommercially available analysis software, robotics, and detectioninstrumentation capable of detecting the signal generated from theassay. Examples of such detectors include, but are not limited to,spectrophotometers, luminometers, fluorimeters, and devices that measureradioisotope decay. Exemplary high-throughput cell-based assays (e.g.,detecting the presence or level of a target protein in a cell) canutilize ArrayScan® VTI HCS Reader or KineticScan® HCS Reader technology(Cellomics Inc., Pittsburgh, Pa.).

Methods for determining the activity of vWF are also known in the artand described herein (e.g., the working examples). See also, e.g.,Horvath et al. (2004) Exp Clin Cardiol 9(10):31-34. Commercial kits arealso available—Instrumentation Laboratory (Bedford, Mass.; cataloguenumber: 0020004700) and Quest Diagnostics (Madison, N.J.).

In some embodiments, the protein expression level (or activity) of atleast two aHUS biomarker proteins (e.g., at least three proteins, atleast four proteins, at least five proteins, at least six proteins, atleast seven proteins, at least eight proteins, at least nine proteins,at least 10 proteins, at least 11 proteins, at least 12 proteins, atleast 13 proteins, at least 14 proteins, at least 15 proteins, at least16 proteins, at least 17 proteins, at least 18 proteins, at least 19proteins, at least 20 proteins, at least 21 proteins, at least 22proteins, at least 23 proteins, or at least 24 proteins or more) can beassessed and/or measured.

In some embodiments, the biological fluid in which the aHUS biomarkerproteins are measured is blood. In some embodiments, the biologicalfluid is a blood fraction, e.g., serum or plasma. In some embodiments,the biological fluid is urine. In some embodiments, all of themeasurements are performed on one biological fluid sample (e.g., a serumsample). In some embodiments, measurements are performed on at least twodifferent biological fluids obtained from the subject. For example, insome embodiments, the concentration or activity of one or more aHUSbiomarker proteins is measured in a serum sample obtained from thepatient. In some embodiments, a blood sample and a urine sample areavailable so as to allow for testing of different analytes in twodifferent sample matrices.

The subject can be, e.g., a human having, suspected of having, or atrisk for developing, aHUS. The subject can be one who has been (or isbeing) treated with an inhibitor of complement (e.g., an inhibitor ofcomplement component C5 such as an anti-C5 antibody). The treatment canhave occurred less than one month (e.g., less than 31, 30, 29, 28, 27,26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9,8, 7, 6, 5, 4, 3, 2, or 1 day) prior to obtaining the sample from thesubject.

The method can further include the step of determining whether thesubject has or is at risk of developing aHUS. Where the subject has beentreated or is being treated with a complement inhibitor (e.g., ananti-C5 antibody) under a predetermined dosing schedule, the method canfurther include determining whether the patient is responsive(therapeutically) to the complement inhibitor therapy.

In some embodiments of any of the methods described herein, the methodrequires recording the measured value(s) of the concentration of the atleast one aHUS biomarker protein. The recordation can be written or on acomputer readable medium. The method can also include communicating themeasured value(s) of the concentration of the at least one aHUSbiomarker protein to the subject and/or to a medical practitioner inwhose care the subject is placed.

In some embodiments, any of the methods described herein can include thestep of administering to the subject the complement inhibitor at ahigher dose or with an increased frequency of dosing, relative to thepredetermined dosing schedule, if the subject is not responsive totreatment with the inhibitor under the predetermined dosing schedule.

Some of the methods described herein involve comparing the measuredconcentration or activity of an aHUS biomarker protein (as measured in abiological sample obtained from a subject) to a control sample. In someembodiments, control sample is obtained from the subject prior toadministering to the subject a complement inhibitor (e.g., a C5inhibitor such as eculizumab). In some embodiments, the control samplecan be (or can be based on), e.g., a collection of samples obtained fromone or more (e.g., two, three, four, five, six, seven, eight, nine, 10,15, 20, 25, 30, 35, or 40 or more) healthy individuals that have notbeen administered a complement inhibitor. In some embodiments, thecontrol sample can be (or can be based on), e.g., a pooled sampleobtained from two or more (e.g., two, three, four, five, six, seven,eight, nine, 10, 15, 20, 25, 30, 35, or 40 or more) individuals. In someembodiments of any of the methods described herein, the pooled samplescan be from healthy individuals, or at least, individuals who do nothave or are not suspected of having (nor at risk for developing) aHUS.For example, determining whether a subject is one having aHUS caninvolve comparing the measured concentration of one or more serumbiomarkers in the subject and comparing the measured concentration tothe average concentration of the same biomarkers in the pooled healthysamples. Similarly, determining whether the concentration or activity ofan aHUS associated biomarker has been reduced following treatment with acomplement inhibitor can involve comparing the concentration or activityof the protein in a biological fluid obtained from a subject prior totreatment with a complement inhibitor to the concentration of protein ina sample of the same biological fluid obtained from the patient aftertreatment with the inhibitor (e.g., one day, two days, three days, fourdays, five days, six days, 1 week, 2 weeks, 3 weeks, a month, 6 weeks,two months, or three months after treatment (e.g., the first of a seriesof treatment in chronic therapy) with the inhibitor).

In some embodiments, determining whether a complement inhibitor hasproduced a desired effect (e.g., a reduction in the concentration oractivity of an aHUS biomarker protein) in a human can be performed byquerying whether the post-treatment concentration of the protein fallswithin a predetermined range indicative of responsiveness to acomplement inhibitor by a human. In some embodiments, determiningwhether a complement inhibitor has produced a desired effect in a humancan include querying if the post-treatment concentration or activity ofone or more aHUS biomarker proteins falls above or below a predeterminedcut-off value. A cut-off value is typically the concentration oractivity of a given protein in a given biological fluid above or belowwhich is considered indicative of a certain phenotype—e.g.,responsiveness to therapy with a complement inhibitor.

In some embodiments of any of the methods described herein, the samepractitioner may administer the complement inhibitor to the subjectprior to determining whether a change in the concentration or activityof one or more aHUS biomarker proteins has occurred, whereas in someembodiments, the practitioner who administers the inhibitor to thesubject is different from the practitioner who determines whether aresponse has occurred in the subject. In some embodiments, thepractitioner may obtain a biological sample (e.g., the blood sample)from the subject prior to administration of the inhibitor. In someembodiments, the practitioner may obtain a biological sample (e.g., ablood sample) from the subject following the administration of theinhibitor to the subject. In some embodiments, the post-treatment samplecan be obtained from the subject less than 48 (e.g., less than 47, 46,45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28,27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10,nine, eight, seven, six, five, four, three, two, or even less than one)hour following administration of the inhibitor to the subject. In someembodiments, the post-treatment sample can be obtained from the subjectless than 20 (e.g., less than 19, 18, 17, 16, 15, 14, 13, 12, 11, 10,nine, eight, seven, six, five, four, three, two, or one) day(s) afteradministering to the subject the inhibitor. In some embodiments, thebiological sample is obtained from the subject no more than 20 (e.g., nomore than 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, nine, eight, seven,six, five, four, three, two, or one) day(s) after the inhibitor isadministered to the subject.

In some embodiments, various steps of the methods described herein canbe performed by more than one practitioner. For example, onepractitioner may analyze (e.g., measure the concentration or activity ofone or more aHUS biomarker proteins in) the pre- and post-treatmentsamples obtained from the subject. Another practitioner may receiveinformation regarding the analysis of the samples by the firstpractitioner to thereby determine whether, e.g., the subject hasresponded to treatment with a complement inhibitor. In some embodiments,yet another practitioner may obtain a pre-treatment biological samplefrom a patient and a fourth practitioner may obtain a post-treatmentbiological sample from the subject. In some embodiments, all steps arecarried out by the same practitioner.

Biological Samples and Sample Collection

Suitable biological samples for use in the methods described hereininclude, e.g., any biological fluid. A biological sample can be, forexample, a specimen obtained from a subject (e.g., a mammal such as ahuman) or can be derived from such a subject. A biological sample canalso be a biological fluid such as urine, whole blood or a fractionthereof (e.g., plasma or serum), saliva, semen, sputum, cerebrospinalfluid, tears, or mucus. A biological sample can be further fractionated,if desired, to a fraction containing particular analytes (e.g.,proteins) of interest. For example, a whole blood sample can befractionated into serum or into fractions containing particular types ofproteins. If desired, a biological sample can be a combination ofdifferent biological samples from a subject such as a combination of twodifferent fluids.

Biological samples suitable for the invention may be fresh or frozensamples collected from a subject, or archival samples with knowndiagnosis, treatment and/or outcome history. The biological samples canbe obtained from a subject, e.g., a subject having, suspected of having,or at risk of developing, a complement-associated disorder such as aHUS.Any suitable methods for obtaining the biological samples can beemployed, although exemplary methods include, e.g., phlebotomy, swab(e.g., buccal swab), lavage, or fine needle aspirate biopsy procedure.Biological samples can also be obtained from bone marrow.

In some embodiments, a protein extract may be prepared from a biologicalsample. In some embodiments, a protein extract contains the totalprotein content. Methods of protein extraction are well known in theart. See, e.g., Roe (2001) “Protein Purification Techniques: A PracticalApproach”, 2^(nd) Edition, Oxford University Press. Numerous differentand versatile kits can be used to extract proteins from bodily fluidsand tissues, and are commercially available from, for example, BioRadLaboratories (Hercules, Calif.), BD Biosciences Clontech (Mountain View,Calif.), Chemicon International, Inc. (Temecula, Calif.), Calbiochem(San Diego, Calif.), Pierce Biotechnology (Rockford, Ill.), andInvitrogen Corp. (Carlsbad, Calif.).

Methods for obtaining and/or storing samples that preserve the activityor integrity of cells in the biological sample are well known to thoseskilled in the art. For example, a biological sample can be furthercontacted with one or more additional agents such as appropriate buffersand/or inhibitors, including protease inhibitors, the agents meant topreserve or minimize changes (e.g., changes in osmolarity or pH) inprotein structure. Such inhibitors include, for example, chelators suchas ethylenediamine tetraacetic acid (EDTA), ethylene glycol tetraaceticacid (EGTA), protease inhibitors such as phenylmethylsulfonyl fluoride(PMSF), aprotinin, and leupeptin. Appropriate buffers and conditions forstoring or otherwise manipulating whole cells are described in, e.g.,Pollard and Walker (1997), “Basic Cell Culture Protocols,” volume 75 ofMethods in molecular biology, Humana Press; Masters (2000) “Animal cellculture: a practical approach,” volume 232 of Practical approach series,Oxford University Press; and Jones (1996) “Human cell cultureprotocols,” volume 2 of Methods in molecular medicine, Humana Press.

A sample also can be processed to eliminate or minimize the presence ofinterfering substances. For example, a biological sample can befractionated or purified to remove one or more materials (e.g., cells)that are not of interest. Methods of fractionating or purifying abiological sample include, but are not limited to, flow cytometry,fluorescence activated cell sorting, and sedimentation.

Complement Inhibitors

Any compound which binds to and inhibits, or otherwise inhibits, thegeneration and/or activity of any of the human complement components maybe utilized in accordance with the present disclosure. For example, aninhibitor of complement can be, e.g., a small molecule, a nucleic acidor nucleic acid analog, a peptidomimetic, or a macromolecule that is nota nucleic acid or a protein. These agents include, but are not limitedto, small organic molecules, RNA aptamers, L-RNA aptamers, Spiegelmers,antisense compounds, double stranded RNA, small interfering RNA, lockednucleic acid inhibitors, and peptide nucleic acid inhibitors. In someembodiments, a complement inhibitor may be a protein or proteinfragment.

In some embodiments, the compositions contain antibodies specific to ahuman complement component. Some compounds include antibodies directedagainst complement components C1, C2, C3, C4, C5, C6, C7, C8, C9, FactorD, Factor B, Factor P, MBL, MASP-1, MASP-2, properdin, or abiologically-active fragment of any of the foregoing, thus preventingthe generation of the anaphylatoxic activity associated with C5a and/orpreventing the assembly of the membrane attack complex C5b-9.

The compositions can also contain naturally occurring or soluble formsof complement inhibitory compounds such as CR1, LEX-CR1, MCP, DAF, CD59,Factor H, cobra venom factor, FUT-175, complestatin, and K76 COOH. Othercompounds which may be utilized to bind to or otherwise block thegeneration and/or activity of any of the human complement componentsinclude, but are not limited to, proteins, protein fragments, peptides,small molecules, RNA aptamers including ARC187 (which is commerciallyavailable from Archemix Corporation, Cambridge, Mass.), L-RNA aptamers,spiegelmers, antisense compounds, serine protease inhibitors, moleculeswhich may be utilized in RNA interference (RNAi) such as double strandedRNA including small interfering RNA (siRNA), locked nucleic acid (LNA)inhibitors, peptide nucleic acid (PNA) inhibitors, etc.

In some embodiments, the complement inhibitor inhibits the activation ofcomplement. For example, the complement inhibitor can bind to andinhibit the complement activation activity of C1 (e.g., C1q, C1r, orC1s) or the complement inhibitor can bind to and inhibit (e.g., inhibitcleavage of) C2, C3, or C4. In some embodiments, the inhibitor inhibitsformation or assembly of the C3 convertase and/or C5 convertase of thealternative and/or classical pathways of complement. In someembodiments, the complement inhibitor inhibits terminal complementformation, e.g., formation of the C5b-9 membrane attack complex. Forexample, an antibody complement inhibitor may include an anti-C5antibody. Such anti-C5 antibodies may directly interact with C5 and/orC5b, so as to inhibit the formation of and/or physiologic function ofC5b.

In some embodiments, the compositions described herein can contain aninhibitor of human complement component C5 (e.g., an antibody, orantigen-binding fragment thereof, that binds to a human complementcomponent C5 protein or a biologically-active fragment thereof such asC5a or C5b). As used herein, an “inhibitor of complement component C5”is any agent that inhibits: (i) the expression, or proper intracellulartrafficking or secretion by a cell, of a complement component C5protein; (ii) the activity of C5 cleavage fragments C5a or C5b (e.g.,the binding of C5a to its cognate cellular receptors or the binding ofC5b to C6 and/or other components of the terminal complement complex;see above); (iii) the cleavage of a human C5 protein to form C5a andC5b; (iv) the proper intracellular trafficking of, or secretion by acell, of a complement component C5 protein; or (v) the stability of C5protein or the mRNA encoding C5 protein. Inhibition of complementcomponent C5 protein expression includes: inhibition of transcription ofa gene encoding a human C5 protein; increased degradation of an mRNAencoding a human C5 protein; inhibition of translation of an mRNAencoding a human C5 protein; increased degradation of a human C5protein; inhibition of proper processing of a pre-pro human C5 protein;or inhibition of proper trafficking or secretion by a cell of a human C5protein. Methods for determining whether a candidate agent is aninhibitor of human complement component C5 are known in the art anddescribed herein.

An inhibitor of human complement component C5 can be, e.g., a smallmolecule, a polypeptide, a polypeptide analog, a nucleic acid, or anucleic acid analog.

“Small molecule” as used herein, is meant to refer to an agent, whichhas a molecular weight preferably of less than about 6 kDa and mostpreferably less than about 2.5 kDa. Many pharmaceutical companies haveextensive libraries of chemical and/or biological mixtures comprisingarrays of small molecules, often fungal, bacterial, or algal extracts,which can be screened with any of the assays of the application. Thisapplication contemplates using, among other things, small chemicallibraries, peptide libraries, or collections of natural products. Tan etal. described a library with over two million synthetic compounds thatis compatible with miniaturized cell-based assays (J Am Chem Soc (1998)120:8565-8566). It is within the scope of this application that such alibrary may be used to screen for agents that bind to a target antigenof interest (e.g., complement component C5). There are numerouscommercially available compound libraries, such as the ChembridgeDIVERSet. Libraries are also available from academic investigators, suchas the Diversity set from the NCI developmental therapeutics program.Rational drug design may also be employed. For example, rational drugdesign can employ the use of crystal or solution structural informationon the human complement component C5 protein. See, e.g., the structuresdescribed in Hagemann et al. (2008) J Biol Chem 283(12):7763-75 andZuiderweg et al. (1989) Biochemistry 28(1):172-85. Rational drug designcan also be achieved based on known compounds, e.g., a known inhibitorof C5 (e.g., an antibody, or antigen-binding fragment thereof, thatbinds to a human complement component C5 protein).

Peptidomimetics can be compounds in which at least a portion of asubject polypeptide is modified, and the three dimensional structure ofthe peptidomimetic remains substantially the same as that of the subjectpolypeptide. Peptidomimetics may be analogues of a subject polypeptideof the disclosure that are, themselves, polypeptides containing one ormore substitutions or other modifications within the subject polypeptidesequence. Alternatively, at least a portion of the subject polypeptidesequence may be replaced with a nonpeptide structure, such that thethree-dimensional structure of the subject polypeptide is substantiallyretained. In other words, one, two or three amino acid residues withinthe subject polypeptide sequence may be replaced by a non-peptidestructure. In addition, other peptide portions of the subjectpolypeptide may, but need not, be replaced with a non-peptide structure.Peptidomimetics (both peptide and non-peptidyl analogues) may haveimproved properties (e.g., decreased proteolysis, increased retention orincreased bioavailability). Peptidomimetics generally have improved oralavailability, which makes them especially suited to treatment ofdisorders in a human or animal. It should be noted that peptidomimeticsmay or may not have similar two-dimensional chemical structures, butshare common three-dimensional structural features and geometry. Eachpeptidomimetic may further have one or more unique additional bindingelements.

Nucleic acid inhibitors can be used to bind to and inhibit a targetantigen of interest. The nucleic acid antagonist can be, e.g., anaptamer. Aptamers are short oligonucleotide sequences that can be usedto recognize and specifically bind almost any molecule, including cellsurface proteins. The systematic evolution of ligands by exponentialenrichment (SELEX) process is powerful and can be used to readilyidentify such aptamers. Aptamers can be made for a wide range ofproteins of importance for therapy and diagnostics, such as growthfactors and cell surface antigens. These oligonucleotides bind theirtargets with similar affinities and specificities as antibodies do (see,e.g., Ulrich (2006) Handb Exp Pharmacol. 173:305-326).

In some embodiments, the complement inhibitor is a non-antibody scaffoldprotein. These proteins are, generally, obtained through combinatorialchemistry-based adaptation of pre-existing antigen-binding proteins. Forexample, the binding site of human transferrin for human transferrinreceptor can be modified using combinatorial chemistry to create adiverse library of transferrin variants, some of which have acquiredaffinity for different antigens. Ali et al. (1999) J Biol Chem274:24066-24073. The portion of human transferrin not involved withbinding the receptor remains unchanged and serves as a scaffold, likeframework regions of antibodies, to present the variant binding sites.The libraries are then screened, as an antibody library is, against atarget antigen of interest to identify those variants having optimalselectivity and affinity for the target antigen. Non-antibody scaffoldproteins, while similar in function to antibodies, are touted as havinga number of advantages as compared to antibodies, which advantagesinclude, among other things, enhanced solubility and tissue penetration,less costly manufacture, and ease of conjugation to other molecules ofinterest. Hey et al. (2005) TRENDS Biotechnol 23(10):514-522.

One of skill in the art would appreciate that the scaffold portion ofthe non-antibody scaffold protein can include, e.g., all or part of: theZ domain of S. aureus protein A, human transferrin, human tenthfibronectin type III domain, kunitz domain of a human trypsin inhibitor,human CTLA-4, an ankyrin repeat protein, a human lipocalin, humancrystallin, human ubiquitin, or a trypsin inhibitor from E. elaterium.Id.

In some embodiments, the complement inhibitor is an antibody, orantigen-binding fragment thereof, which binds to a human complementcomponent C5 protein. (Hereinafter, the antibody may sometimes bereferred to as an “anti-C5 antibody.”)

In some embodiments, the anti-C5 antibody can bind to an epitope in thealpha chain of the human complement component C5 protein. Antibodiesthat bind to the alpha chain of C5 are described in, for example, PCTapplication publication no. WO 2010/015608 and U.S. Pat. No. 6,355,245.In some embodiments, the anti-C5 antibody can bind to an epitope in thebeta chain of the human complement component C5 protein. Antibodies thatbind to the C5 beta chain are described in, e.g., Moongkarndi et al.(1982) Immunobiol 162:397; Moongkarndi et al. (1983) Immunobiol 165:323;and Mollnes et al. (1988) Scand J Immunol 28:307-312.

Additional exemplary antigenic fragments of human complement componentC5 are disclosed in, e.g., U.S. Pat. No. 6,355,245, the disclosure ofwhich is incorporated herein by reference.

Additional anti-C5 antibodies, and antigen-binding fragments thereof,suitable for use in the fusion proteins described herein are describedin, e.g., PCT application publication no. WO 2010/015608, the disclosureof which is incorporated herein by reference in its entirety.

In some embodiments, the anti-C5 antibody specifically binds to a humancomplement component C5 protein (e.g., the human C5 protein having theamino acid sequence depicted in SEQ ID NO:1). The terms “specificbinding” or “specifically binds” refer to two molecules forming acomplex (e.g., a complex between an antibody and a complement componentC5 protein) that is relatively stable under physiologic conditions.Typically, binding is considered specific when the association constant(K_(a)) is higher than 10⁶ M⁻¹. Thus, an antibody can specifically bindto a C5 protein with a K_(a) of at least (or greater than) 10⁶ (e.g., atleast or greater than 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹10¹², 10¹³, 10¹⁴, or 10¹⁵or higher) M. Examples of antibodies that specifically bind to a humancomplement component C5 protein are described in, e.g., U.S. Pat. No.6,355,245, the disclosure of which is incorporated herein by referencein its entirety.

The anti-C5 antibodies described herein can have activity in blockingthe generation or activity of the C5a and/or C5b active fragments of acomplement component C5 protein (e.g., a human C5 protein). Through thisblocking effect, the anti-C5 antibodies inhibit, e.g., theproinflammatory effects of C5a and the generation of the C5b-9 membraneattack complex (MAC) at the surface of a cell. Anti-05 antibodies thathave the ability to block the generation of C5a are described in, e.g.,Moongkarndi et al. (1982) Immunobiol 162:397 and Moongkarndi et al.(1983) Immunobiol 165:323.

In some embodiments, an anti-C5 antibody, or antigen-binding fragmentthereof, can reduce the ability of a C5 protein to bind to humancomplement component C3b (e.g., C3b present in an AP or CP C5 convertasecomplex) by greater than 50 (e.g., greater than 55, 60, 65, 70, 75, 80,85, 90, or 95 or more) %. In some embodiments, upon binding to a C5protein, the anti-C5 antibody or antigen-binding fragment thereof canreduce the ability of the C5 protein to bind to complement component C4b(e.g., C4b present in a CP C5 convertase) by greater than 50 (e.g.,greater than 55, 60, 65, 70, 75, 80, 85, 90, or 95 or more) %. Methodsfor determining whether an antibody can block the generation or activityof the C5a and/or C5b active fragments of a complement component C5protein, or binding to complement component C4b or C3b, are known in theart and described in, e.g., U.S. Pat. No. 6,355,245 and Wurzner et al.(1991) Complement Inflamm 8:328-340.

In some embodiments, the composition comprises, and/or the antibody is,eculizumab (Soliris®; Alexion Pharmaceuticals, Inc., Cheshire, Conn.).(See, e.g., Kaplan (2002) Curr Opin Investig Drugs 3(7):1017-23; Hill(2005) Clin Adv Hematol Oncol 3(11):849-50; and Rother et al. (2007)Nature Biotechnology 25(11):1256-1488.) In some embodiments, thecomposition comprises, and/or the antibody is, pexelizumab (AlexionPharmaceuticals, Inc., Cheshire, Conn.). (See, e.g., Whiss (2002) CurrOpin Investig Drugs 3(6):870-7; Patel et al. (2005) Drugs Today (Barc)41(3):165-70; and Thomas et al. (1996) Mol Immunol 33(17-18):1389-401.)

In some embodiments, the C5 inhibitor is an antibody that binds to C5a(sometimes referred to herein as “an anti-C5a antibody”). In someembodiments, the antibody binds to C5a, but not to full-length C5. Insome embodiments, the binding of an antibody to C5a can inhibit thebiological activity of C5a. Methods for measuring C5a activity include,e.g., chemotaxis assays, RIAs, or ELISAs (see, e.g., Ward and Zvaifler(1971) J Clin Invest 50(3):606-16 and Wurzner et al. (1991) ComplementInflamm 8:328-340). In some embodiments, the binding of an antibody toC5a can inhibit the interaction between C5a and C5aR1. Suitable methodsfor detecting and/or measuring the interaction between C5a and C5aR1 (inthe presence and absence of an antibody) are known in the art anddescribed in, e.g., Mary and Boulay (1993) Eur J Haematol 51(5):282-287;Kaneko et al. (1995) Immunology 86(1):149-154; Giannini et al. (1995) JBiol Chem 270(32):19166-19172; and U.S. Patent Application PublicationNo. 20060160726. For example, the binding of detectably labeled (e.g.,radioactively labeled) C5a to C5aR1-expressing peripheral bloodmononuclear cells can be evaluated in the presence and absence of anantibody. A decrease in the amount of detectably-labeled C5a that bindsto C5aR1 in the presence of the antibody, as compared to the amount ofbinding in the absence of the antibody, is an indication that theantibody inhibits the interaction between C5a and C5aR1. In someembodiments, the binding of an antibody to C5a can inhibit theinteraction between C5a and C5L2 (see below). Methods for detectingand/or measuring the interaction between C5a and C5L2 are known in theart and described in, e.g., Ward (2009) J Mol Med 87(4):375-378 and Chenet al. (2007) Nature 446(7132):203-207 (see below).

In some embodiments, the C5 inhibitor is an antibody that binds to C5b(sometimes referred to herein as “an anti-C5b antibody”). In someembodiments, the antibody binds to C5b, but does not bind to full-lengthC5. The structure of C5b is described in, e.g., Müller-Eberhard (1985)Biochem Soc Symp 50:235-246; and Yamamoto and Gewurz (1978) J Immunol120(6):2008-2015. As described above, C5b combines with C6, C7, and C8to form the C5b-8 complex at the surface of the target cell. Proteincomplex intermediates formed during the series of combinations includeC5b-6 (including C5b and C6), C5b-7 (including C5b, C6, and C7), andC5b-8 (including C5b, C6, C7, and C8). Upon binding of several C9molecules, the membrane attack complex (MAC, C5b-9 terminal complementcomplex (TCC)) is formed. When sufficient numbers of MACs insert intotarget cell membranes, the openings they create (MAC pores) mediaterapid osmotic lysis of the target cells.

In some embodiments, the binding of an antibody to C5b can inhibit theinteraction between C5b and C6. In some embodiments, the binding of theantibody to C5b can inhibit the assembly or activity of the C5b-9MAC-TCC. In some embodiments, the binding of an antibody to C5b caninhibit complement-dependent cell lysis (e.g., in vitro and/or in vivo).Suitable methods for evaluating whether an antibody inhibitscomplement-dependent lysis include, e.g., hemolytic assays or otherfunctional assays for detecting the activity of soluble C5b-9. Forexample, a reduction in the cell-lysing ability of complement in thepresence of an antibody can be measured by a hemolysis assay describedby Kabat and Mayer (eds.), “Experimental Immunochemistry, 2^(nd)Edition,” 135-240, Springfield, Ill., CC Thomas (1961), pages 135-139,or a conventional variation of that assay such as the chickenerythrocyte hemolysis method as described in, e.g., Hillmen et al.(2004) N Engl J Med 350(6):552.

Antibodies that bind to C5b as well as methods for making suchantibodies are known in the art. Commercially available anti-C5bantibodies are available from a number of vendors including, e.g.,Hycult Biotechnology (catalogue number: HM2080; clone 568) and Abcam™(ab46151 or ab46168).

Methods for determining whether a particular agent is an inhibitor ofhuman complement component C5 are described herein and are known in theart. For example, the concentration and/or physiologic activity of C5aand C5b in a body fluid can be measured by methods well known in theart. Methods for measuring C5a concentration or activity include, e.g.,chemotaxis assays, RIAs, or ELISAs (see, e.g., Ward and Zvaifler (1971)J Clin Invest. 50(3):606-16 and Wurzner et al. (1991) ComplementInflamm. 8:328-340). For C5b, hemolytic assays or assays for solubleC5b-9 as discussed herein can be used. Other assays known in the art canalso be used. Using assays of these or other suitable types, candidateagents capable of inhibiting human complement component C5 such as ananti-C5 antibody, can be screened in order to, e.g., identify compoundsthat are useful in the methods described herein and determine theappropriate dosage levels of such compounds.

Methods for determining whether a candidate compound inhibits thecleavage of human C5 into forms C5a and C5b are known in the art anddescribed in, e.g., Moongkarndi et al. (1982) Immunobiol 162:397;Moongkarndi et al. (1983) Immunobiol 165:323; Isenman et al. (1980) JImmunol 124(1):326-31; Thomas et al. (1996) Mol. Immunol33(17-18):1389-401; and Evans et al. (1995) Mol. Immunol 32(16):1183-95.

Inhibition of human complement component C5 can also reduce thecell-lysing ability of complement in a subject's body fluids. Suchreductions of the cell-lysing ability of complement present can bemeasured by methods well known in the art such as, for example, by aconventional hemolytic assay such as the hemolysis assay described byKabat and Mayer (eds), “Experimental Immunochemistry, 2^(nd) Edition,”135-240, Springfield, Ill., CC Thomas (1961), pages 135-139, or aconventional variation of that assay such as the chicken erythrocytehemolysis method as described in, e.g., Hillmen et al. (2004) N Engl JMed 350(6):552.

Antibodies that bind to C3b and, for example, inhibit the C3b convertaseare also well known in the art. See for example, PCT applicationpublication nos. WO 2010/136311, WO 2009/056631, and WO 2008/154251, thedisclosures of each of which are incorporated herein by reference intheir entirety. Antagonistic anti-C6 and anti-C7 antibodies have beendescribed in, e.g., Brauer et al. (1996) Transplantation 61(4):588-594and U.S. Pat. No. 5,679,345.

In some embodiments, the antibody is an anti-factor B antibody (such asthe monoclonal antibody 1379 produced by ATCC Deposit No. PTA-6230).Anti-factor B antibodies are also described in, e.g., Ueda et al. (1987)J Immunol 138(4):1143-9; Tanhehco et al. (1999) Transplant Proc31(5):2168-71; U.S. Pat. Nos. 7,999,082 and 7,964,705; and PCTpublication no. WO 09/029669.

In some embodiments, the antibody is an anti-factor D antibody, e.g., anantibody described in Pascual et al. (1990) J Immunol Methods127:263-269; Sahu et al. (1993) Mol Immunol 30(7):679-684; Pascual etal. (1993) Eur J Immunol 23:1389-1392; Niemann et al. (1984) J Immunol132(2):809-815; U.S. Pat. No. 7,439,331; or U.S. patent applicationpublication no. 20080118506.

In some embodiments, the antibody is an anti-properdin antibody.Suitable anti-properdin antibodies are also well-known in the art andinclude, e.g., U.S. patent application publication nos. 20110014614 andPCT application publication no. WO2009110918.

Methods for Treatment

Also provided herein are compositions and methods for treating orpreventing aHUS in a subject (e.g., a human). The compositions (e.g.,complement inhibitors and/or secondary agents) can be administered to asubject, e.g., a human subject, using a variety of methods that depend,in part, on the route of administration. The route can be, e.g.,intravenous injection or infusion (IV), subcutaneous injection (SC),intraperitoneal (IP) injection, or intramuscular injection.

Administration can be achieved by, e.g., local infusion, injection, orby means of an implant. The implant can be of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. The implant can be configured for sustained or periodicrelease of the composition to the subject. See, e.g., U.S. patentpublication no. 20080241223; U.S. Pat. Nos. 5,501,856; 4,863,457; and3,710,795; and European patent nos. EP488401 and EP430539, thedisclosures of each of which are incorporated herein by reference intheir entirety. The composition can be delivered to the subject by wayof an implantable device based on, e.g., diffusive, erodible orconvective systems, e.g., osmotic pumps, biodegradable implants,electrodiffusion systems, electroosmosis systems, vapor pressure pumps,electrolytic pumps, effervescent pumps, piezoelectric pumps,erosion-based systems, or electromechanical systems.

A suitable dose of a complement inhibitor (e.g., an anti-C5 antibody orfragment thereof), which dose is capable of treating or preventing aHUSin a subject, can depend on a variety of factors including, e.g., theage, sex, and weight of a subject to be treated and the particularinhibitor compound used. For example, a different dose of an siRNAspecific for human C5 may be required to treat a subject with aHUS ascompared to the dose of an anti-C5 antibody required to treat the samepatient. Other factors affecting the dose administered to the subjectinclude, e.g., the type or severity of the aHUS. For example, a subjecthaving CFH-associated aHUS may require administration of a differentdosage of the inhibitor than a subject with MCP-associated aHUS. Otherfactors can include, e.g., other medical disorders concurrently orpreviously affecting the subject, the general health of the subject, thegenetic disposition of the subject, diet, time of administration, rateof excretion, drug combination, and any other additional therapeuticsthat are administered to the subject. It should also be understood thata specific dosage and treatment regimen for any particular subject willdepend upon the judgment of the treating medical practitioner (e.g.,doctor or nurse).

The inhibitor can be administered as a fixed dose, or in a milligram perkilogram “mg/kg” dose. In some embodiments, the dose can also be chosento reduce or avoid production of antibodies or other host immuneresponses against one or more active agents in the composition. While inno way intended to be limiting, exemplary dosages of an inhibitor, suchas an anti-C5 antibody, include, e.g., 1-100 mg/kg, 0.5-50 mg/kg,0.1-100 mg/kg, 0.5-25 mg/kg, 1-20 mg/kg, and 1-10 mg/kg of body weight.

In some embodiments, a human can be intravenously administered ananti-C5 antibody (e.g., eculizumab) at a dose of about 900 mg aboutevery 12 (e.g., about every 10, 11, 13, 14, 15, 16, 17, 18, 19, 20, 21,28, 30, 42, or 49 or more) days. See, e.g., Hill et al. (2005) Blood106(7):2559.

In some embodiments, a human can be intravenously administered ananti-C5 antibody (e.g., eculizumab) at a dose of about 600 (e.g., about625, 650, 700, 725, 750, 800, 825, 850, 875, 900, 925, 950, or 1,000 ormore) mg every week, optionally, for two or more (e.g., three, four,five, six, seven, or eight or more) weeks. Following the initialtreatment, the human can be administered the antibody at a dose of about900 mg about every 14 (e.g., about every 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 28, 30, 42, or 49 or more) days, e.g., as a maintenancedose. See, e.g., Hillmen et al. (2004) N Engl J Med. 350(6):552-9 andDmytrijuk et al. (2008) The Oncologist 13(9):993.

In some embodiments, a human can be intravenously administered ananti-C5 antibody (e.g., eculizumab) at a dose of about 900 (e.g., 925,950, 975, 1000, 1100, or 1200 or more) mg every week, optionally, fortwo or more (e.g., three, four, five, six, seven, or eight or more)weeks. Following the initial treatment, the human can be administeredthe antibody at a dose of about 1200 mg about every 14 (e.g., aboutevery 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 28, 30, 42, or 49 ormore) days, e.g., as a maintenance dose. See, e.g., International patentapplication publication no. WO 2010/054403.

As used herein, “chronically administered,” “chronic treatment,”“treating chronically,” or similar grammatical variations thereof referto a treatment regimen that is employed to maintain a certain thresholdconcentration of a therapeutic agent in the blood of a patient in orderto completely or substantially suppress systemic complement activity inthe patient over a prolonged period of time. Accordingly, a patientchronically treated with a complement inhibitor can be treated for aperiod of time that is greater than or equal to 2 weeks (e.g., 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 weeks; 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, or 12 months; or 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6,6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, or 12 years or for the remainderof the patient's life) with the inhibitor in an amount and with a dosingfrequency that are sufficient to maintain a concentration of theinhibitor in the patient's blood that inhibits or substantially inhibitssystemic complement activity in the patient. In some embodiments, thecomplement inhibitor can be chronically administered to a patient inneed thereof in an amount and with a frequency that are effective tomaintain serum hemolytic activity at less than or equal to 20 (e.g., 19,18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5) %. See, e.g., Hillet al. (2005) Blood 106(7):2559. In some embodiments, the complementinhibitor can be administered to a patient in an amount and with afrequency that are effective to maintain serum lactate dehydrogenase(LDH) levels at within at least 20 (e.g., 19, 18, 17, 16, 15, 14, 13,12, 11, 10, 9, 8, 7, 6, or 5) % of the normal range for LDH. See Hill etal. (2005) supra. In some embodiments, the complement inhibitor isadministered to the patient in an amount and with a frequency that areeffective to maintain a serum LDH level less than 550 (e.g., less than540, 530, 520, 510, 500, 490, 480, 470, 460, 450, 440, 430, 420, 410,400, 390, 380, 370, 360, 350, 340, 330, 320, 310, 300, 290, 280, or lessthan 270) IU/L. To maintain systemic complement inhibition in a patient,the complement inhibitor can be chronically administered to the patient,e.g., once a week, once every two weeks, twice a week, once a day, oncea month, or once every three weeks.

A pharmaceutical composition can include a therapeutically effectiveamount of an inhibitor of human complement component C5 (e.g., ananti-C5 antibody or antigen-binding fragment thereof). Such effectiveamounts can be readily determined by one of ordinary skill in the artbased, in part, on the effect of the administered inhibitor, or thecombinatorial effect of the antibody and one or more additional activeagents, if more than one agent is used. A therapeutically effectiveamount of an inhibitor of human complement component C5 (e.g., ananti-C5 antibody) can also vary according to factors such as the diseasestate, age, sex, and weight of the individual, and the ability of theantibody (and one or more additional active agents) to elicit a desiredresponse in the individual, e.g., amelioration of at least one conditionparameter, e.g., amelioration of at least one symptom of aHUS. Forexample, a therapeutically effective amount of an inhibitor of humancomplement component C5 (e.g., an anti-C5 antibody) can inhibit (lessenthe severity of or eliminate the occurrence of) and/or preventthrombocytopenia, microangiopathic hemolytic anemia, renal failure,and/or any one of the symptoms of aHUS known in the art or describedherein. A therapeutically effective amount is also one in which anytoxic or detrimental effects of the composition are outweighed by thetherapeutically beneficial effects.

The terms “therapeutically effective amount” or “therapeuticallyeffective dose,” or similar terms used herein are intended to mean anamount of an agent (e.g., an inhibitor of human complement component 5)that will elicit the desired biological or medical response (e.g., animprovement in one or more symptoms of aHUS). In some embodiments, acomposition described herein contains a therapeutically effective amountof an inhibitor of human complement component C5. In some embodiments, acomposition described herein contains a therapeutically effective amountof an antibody, or antigen-binding fragment thereof, which binds to acomplement component C5 protein. In some embodiments, the compositioncontains two or more (e.g., three, four, five, six, seven, eight, nine,10, or 11 or more) different inhibitors of human complement component C5such that the composition as a whole is therapeutically effective. Forexample, a composition can contain an antibody that binds to a human C5protein and an siRNA that binds to, and promotes the degradation of, anmRNA encoding a human C5 protein, wherein the antibody and siRNA areeach at a concentration that when combined are therapeuticallyeffective. In some embodiments, the composition contains the inhibitorand one or more second active agents such that the composition as awhole is therapeutically effective. For example, the composition cancontain an antibody that binds to a human C5 protein and another agentuseful for treating or preventing aHUS.

Toxicity and therapeutic efficacy of such compositions can be determinedby known pharmaceutical procedures in cell cultures or experimentalanimals (animal models of aHUS). These procedures can be used, e.g., fordetermining the LD₅₀ (the dose lethal to 50% of the population) and theED₅₀ (the dose therapeutically effective in 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex and it can be expressed as the ratio LD₅₀/ED₅₀. Compositions, orinhibitors (e.g., anti-C5 antibodies) of the compositions, that exhibithigh therapeutic indices are preferred. While compositions that exhibittoxic side effects may be used, care should be taken to design adelivery system that targets such compounds to the site of affectedtissue and to minimize potential damage to normal cells and, thereby,reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. Suitable animalmodels of aHUS are known in the art and are described in, e.g., Atkinsonet al. (2007) Journal of Experimental Medicine 204(6):1245-1248. Thedosage of such inhibitors lies generally within a range of circulatingconcentrations of the inhibitors (e.g., an anti-C5 antibody orantigen-binding fragment thereof) that include the ED₅₀ with little orno toxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For aninhibitor of human complement component C5 (e.g., an anti-C5 antibody)used as described herein (e.g., for treating or preventing aHUS), thetherapeutically effective dose can be estimated initially from cellculture assays. A dose can be formulated in animal models to achieve acirculating plasma concentration range that includes the IC₅₀ (i.e., theconcentration of the test compound which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma may be measured, for example, by high performance liquidchromatography.

In some embodiments, the required dose of an inhibitor of humancomplement component C5 can be determined based on the concentration ofhuman C5 protein in the subject's blood. For example, a subject having ahigher concentration of circulating human C5 protein levels may requirea higher dose of a human C5 inhibitor than a subject having lower levelsof circulating human C5. Methods for determining the concentration ofhuman complement component C5 in a blood-derived fluid sample from asubject are known in the art and described in, e.g., Rawal et al. (1998)J Biol Chem 273(27):16828-16835.

In some embodiments, the methods can be performed in conjunction withother therapies for aHUS. For example, the composition can beadministered to a subject at the same time, prior to, or after,nephrectomy (e.g., bilateral nephrectomy), dialysis, a plasma exchange,or a plasma infusion (see, e.g., Noris et al. (2005) “Non-shigatoxin-associated hemolytic uremic syndrome.” In: Zipfel P (ed).Complement and Kidney Disease. Basel: Birkhauser-Verlag, 65-83).

A “subject,” as used herein, can be any mammal. For example, a subjectcan be a human, a non-human primate (e.g., monkey, baboon, orchimpanzee), a horse, a cow, a pig, a sheep, a goat, a dog, a cat, arabbit, a guinea pig, a gerbil, a hamster, a rat, or a mouse. In someembodiments, the subject is an infant (e.g., a human infant).

As used herein, a subject “in need of prevention,” “in need oftreatment,” or “in need thereof,” refers to one, who by the judgment ofan appropriate medical practitioner (e.g., a doctor, a nurse, or a nursepractitioner in the case of humans; a veterinarian in the case ofnon-human mammals), would reasonably benefit from a given treatment(such as treatment with a composition comprising an inhibitor of humancomplement component C5).

As used herein, a subject “at risk for developing aHUS” is a subjecthaving one or more (e.g., two, three, four, five, six, seven, or eightor more) risk factors for developing the disorder. Risk factors for aHUSare well known in the art of medicine and include, e.g., apredisposition to develop the condition, i.e., a family history of thecondition or a genetic predisposition to develop the condition such as,e.g., one or more mutations in complement Factor H (CFH), membranecofactor protein (MCP; CD46), C4b-binding protein, complement factor B(CFB), or complement factor I (CFI). See, e.g., Warwicker et al. (1998)Kidney Int 53:836-844; Richards et al. (2001) Am J Hum Genet 68:485-490;Caprioli et al. (2001) Am Soc Nephrol 12:297-307; Neuman et al. (2003) JMed Genet 40:676-681; Richards et al. (2006) Proc Natl Acad Sci USA100:12966-12971; Fremeaux-Bacchi et al. (2005) JAm Soc Nephrol17:2017-2025; Esparza-Gordillo et al. (2005) Hum Mol Genet 14:703-712;Goicoechea de Jorge et al. (2007) Proc Natl Acad Sci USA 104(1):240-245;Blom et al. (2008) J Immunol 180(9):6385-91; and Fremeaux-Bacchi et al.(2004) J Medical Genet 41:e84). See also Kavanagh et al. (2006), supra.Risk factors also include, e.g., infection with Streptococcuspneumoniae, pregnancy, cancer, exposure to anti-cancer agents (e.g.,quinine, mitomycin C, cisplatin, or bleomycin), exposure toimmunotherapeutic agents (e.g., cyclosporine, OKT3, or interferon),exposure to anti-platelet agents (e.g., ticlopidine or clopidogrel), HIVinfection, transplantation, autoimmune disease, and combinedmethylmalonic aciduria and homocystinuria (cb1C). See, e.g.,Constantinescu et al. (2004) Am J Kidney Dis 43:976-982; George (2003)Curr Opin Hematol 10:339-344; Gottschall et al. (1994) Am J Hematol47:283-289; Valavaara et al. (1985) Cancer 55:47-50; Miralbell et al.(1996) J Clin Oncol 14:579-585; Dragon-Durey et al. (2005) J Am SocNephrol 16:555-63; and Becker et al. (2004) Clin Infect Dis39:S267-S275. Thus, a human at risk for developing aHUS can be, e.g.,one who has a family history of aHUS and/or one who has an HIVinfection. From the above it will be clear that subjects “at risk fordeveloping aHUS” are not all the subjects within a species of interest.

A subject “suspected of having aHUS” is one having one or more symptomsof the condition. Symptoms of this condition are well-known to those ofskill in the art of medicine and include, e.g., severe hypertension,proteinuria, uremia, lethargy/fatigue, irritability, thrombocytopenia,microangiopathic hemolytic anemia, and renal function impairment (e.g.,acute renal failure). It will be clear from the foregoing passage thatsubjects “suspected of having aHUS” are not all the subjects within aspecies of interest.

aHUS can be genetic, acquired, or idiopathic. aHUS can be consideredgenetic when two or more (e.g., three, four, five, or six or more)members of the same family are affected by the disease at least sixmonths apart and exposure to a common triggering agent has beenexcluded, or when one or more aHUS-associated gene mutations (e.g., oneor more mutations in CFH, MCP/CD46, CFB, or CFI) are identified in asubject. For example, a subject can have CFH-associated aHUS,CFB-associated aHUS, CFI-associated aHUS, or MCP-associated aHUS. Up to30% of genetic aHUS is associated with mutations in CFH, 12% withmutations in MCP, 5-10% with mutations in CFI, and less than 2% withmutations in CFB. Genetic aHUS can be multiplex (i.e., familial; two ormore affected family members) or simplex (i.e., a single occurrence in afamily). aHUS can be considered acquired when an underlyingenvironmental factor (e.g., a drug, systemic disease, or viral orbacterial agents that do not result in Shiga-like exotoxins) can beidentified. aHUS can be considered idiopathic when no trigger (geneticor environmental) is evident.

In some embodiments, the methods can include identifying the subject asone having, suspected of having, or at risk for developing aHUS. Inaddition to use of the aHUS biomarker profiling described herein,laboratory tests can be performed to determine whether a human subjecthas thrombocytopenia, microangiopathic hemolytic anemia, or acute renalinsufficiency. Thrombocytopenia can be diagnosed by a medicalprofessional as one or more of: (i) a platelet count that is less than150,000/mm³ (e.g., less than 60,000/mm³); (ii) a reduction in plateletsurvival time that is reduced, reflecting enhanced platelet disruptionin the circulation; and (iii) giant platelets observed in a peripheralsmear, which is consistent with secondary activation ofthrombocytopoiesis. Microangiopathic hemolytic anemia can be diagnosedby a medical professional as one or more of: (i) hemoglobinconcentrations that are less than 10 mg/dL (e.g., less than 6.5 mg/dL);(ii) increased serum lactate dehydrogenase (LDH) concentrations (>460U/L); (iii) hyperbilirubinemia, reticulocytosis, circulating freehemoglobin, and low or undetectable haptoglobin concentrations; and (iv)the detection of fragmented red blood cells (schistocytes) with thetypical aspect of burr or helmet cells in the peripheral smear togetherwith a negative Coombs test. See, e.g., Kaplan et al. (1992) “HemolyticUremic Syndrome and Thrombotic Thrombocytopenic Purpura,” Informa HealthCare (ISBN 0824786637) and Zipfel (2005) “Complement and KidneyDisease,” Springer (ISBN 3764371668).

Blood concentrations of C3 and C4 can also be used as a measure ofcomplement activation or dysregulation. In addition, a subject'scondition can be further characterized by identifying the subject asharboring one or more mutations in a gene associated with aHUS such asCFI, CFB, CFH, or MCP (supra). Suitable methods for detecting a mutationin a gene include, e.g., DNA sequencing and nucleic acid arraytechniques. See, e.g., Breslin et al. (2006) Clin Am Soc Nephrol 1:88-99and Goicoechea de Jorge et al. (2007) Proc Natl Acad Sci USA104:240-245.

In some embodiments, the inhibitor of human complement component C5(e.g., an anti-C5 antibody or antigen-binding fragment thereof) can beadministered to a subject as a monotherapy. Alternatively, as describedabove, the inhibitor can be administered to a subject as a combinationtherapy with another treatment, e.g., another treatment for aHUS. Forexample, the combination therapy can include administering to thesubject (e.g., a human patient) one or more additional agents (e.g.,anti-hypertensives) that provide a therapeutic benefit to the subjectwho has, or is at risk of developing, aHUS. In some embodiments, theinhibitor of human complement component C5 and the one or moreadditional active agents are administered at the same time. In otherembodiments, the inhibitor is administered first in time and the one ormore additional active agents are administered second in time. In someembodiments, the one or more additional active agents are administeredfirst in time and the inhibitor is administered second in time.

The inhibitor of human complement component C5 can replace or augment apreviously or currently administered therapy. For example, upon treatingwith an anti-C5 antibody or antigen-binding fragment thereof,administration of the one or more additional active agents can cease ordiminish, e.g., be administered at lower levels. In some embodiments,administration of the previous therapy can be maintained. In someembodiments, a previous therapy will be maintained until the level ofinhibitor of human C5 reaches a level sufficient to provide atherapeutic effect. The two therapies can be administered incombination.

Monitoring a subject (e.g., a human patient) for an improvement in aHUS,as defined herein, means evaluating the subject for a change in adisease parameter, e.g., an improvement in one or more symptoms of thedisease. Such symptoms include any of the symptoms of aHUS describedherein. In some embodiments, the evaluation is performed at least 1hour, e.g., at least 2, 4, 6, 8, 12, 24, or 48 hours, or at least 1 day,2 days, 4 days, 10 days, 13 days, 20 days or more, or at least 1 week, 2weeks, 4 weeks, 10 weeks, 13 weeks, 20 weeks or more, after treatmentbegins. The subject can be evaluated in one or more of the followingperiods: prior to beginning of treatment; during the treatment; or afterone or more elements of the treatment have been administered. Evaluatingcan include evaluating the need for further treatment, e.g., evaluatingwhether a dosage, frequency of administration, or duration of treatmentshould be altered. It can also include evaluating the need to add ordrop a selected therapeutic modality, e.g., adding or dropping any ofthe treatments for aHUS described herein.

Kits

Also provided are kits comprising various reagents and materials usefulfor carrying out the methods described herein. The procedures formeasuring, diagnosing, evaluating, and/or assessing described herein maybe performed by diagnostic laboratories, experimental laboratories, orindividual practitioners. The invention provides kits which can be usedin any or all of these settings.

In some embodiments, the kits described herein comprise materials andreagents for, among other things, characterizing or processingbiological samples (e.g., biological fluids), measuring biomarker levels(e.g., protein or nucleic acid levels), diagnosing aHUS in a subject, ormonitoring treatment response in a subject according to the methodsprovided herein. In certain embodiments, an inventive kit comprises atleast one or more reagents that specifically detect protein levels ofone or more aHUS biomarker proteins (e.g., those selected from Table 1)and, optionally, instructions for using the kit. The kit can include,e.g., any of the arrays described herein.

In some embodiments, the kits may include suitable control samples(e.g., biological fluids from normal healthy individuals or a solutioncomprising a known, control amount of a particular analyte of interest).In some embodiments, kits of the invention may include instructions forusing the kit according to one or more methods described herein and maycomprise instructions for processing the biological sample (e.g., abiological fluid) obtained from the subject and/or for performing thetest or instructions for interpreting the results.

The following examples are intended to illustrate, not limit, theinvention.

EXAMPLES

To better understand the pathology of aHUS, the inventors have collectedsamples of biological fluids (whole blood, serum, plasma, and urine)from patients having aHUS or suspected of having aHUS both before and,at several points, after initiating treatment with a complementinhibitor (the anti-C5 antibody eculizumab). One objective of this studywas to define a series of clinically definable parameters that could beused to monitor responsiveness of patients to treatment with thecomplement inhibitor as well as markers of the disease and progressionor abatement thereof. The inventors identified several proteins whoseexpression and/or activity was correlated with either the aHUS diseasestate and/or responsiveness of an aHUS patient to treatment with acomplement inhibitor. The proteins were those involved or associatedwith complement and/or endothelial cell activation, inflammation, renalinjury, and coagulation (see Table 1).

For the study, a total of 41 adult subjects (27 females and 12 males)with a confirmed diagnosis of aHUS were recruited as were normal healthyadult volunteers. All patients had confirmed aHUS at screening based onone or more of the following characteristics: platelet count less than150×10⁹/L; hemoglobin levels at less than the lower limit of normal; LDHlevels that were greater than or equal to 1.5 times the upper limit ofnormal; serum creatinine levels that were greater than or equal to theupper limit of normal; and an ADAMTS13 activity level that was greaterthan 5%. All patients tested negative for Shiga toxin.

The mean patient age at inclusion was 40.3 years old. 68% of thepatients were female; 2 (4.8%) were black or African-American; and 1patient (2.4%) was of Asian descent. Six patients (14.6%) reported afamily history of aHUS. Twenty (48.7%) had at least one identifiedcomplement regulatory protein mutation or tested positive for anautoantibody that binds to a complement regulatory protein. Thirtypatients (73.2%) presented with a first clinical manifestation of aHUS.Six patients (14%) immediately initiated eculizumab without use ofplasma exchange/infusion (PE/PI). Twenty-four patients (58.5%) were ondialysis at baseline (prior to eculizumab treatment). Nine patients(22%) had previously undergone a renal transplant. Twenty-seven (66%)had a platelet count that was less than 150×10⁹/L. Thirty-two (78%)patients had a serum LDH level that was greater than the upper limit ofnormal. The mean haptoglobin (Hp) levels for the patients in this cohortwas 0.6±0.4 g/L; whereas the mean serum creatinine levels in thispatient cohort was 411±264.6 μmol/L (N=40).

Biological fluids were collected at enrollment in the study (prior totreatment) and then following treatment at each administration of thedrug. Eculizumab was administered to the subjects under the followingschedule: 900 mg once per week for four weeks; 1200 mg as the fifthdose; and 1200 mg once every two weeks thereafter for up to 55 weeks aspart of a Phase 2 clinical trial.

Example 1 Materials and Methods

Urine Samples

Freshly collected urine was immediately mixed with protease inhibitors.The concentrations of several analytes including NGAL, cystatin C,clusterin, TIMP-1, β2-microglobulin, C5b9, C5a, and creatinine in urinecollected from the subjects were measured using commercially-availablekits as described briefly below.

NGAL levels were measured in urine using a commercially available kit(R&D Systems, Minneapolis, Minn.; catalogue number: DLCN20). Briefly,urine samples were diluted 1:3 using kit supplied calibrator diluentRD5-24. 50 μL of each sample or kit standard control (NS0-expressedrecombinant human Lipocalin-2) were added to wells of an assay plate induplicate, each well containing 100 μL of kit-supplied Assay DiluentRD1-52. After a two hour incubation at 4° C. in the refrigerator, wellswere washed four times with 200 μL per well of wash solution. Anenzymatically (horseradish peroxidase)-labeled anti-NGAL conjugate wasadded at 200 μL per well and incubated for two hours at 4° C. in therefrigerator. Wells were washed four times with 200 μL per well of washsolution and developed by adding 200 μL per well of kit-supplied TMBSubstrate Solution (substrate for the enzyme of the anti-NGAL conjugate)and incubated at room temperature in the dark for 30 minutes. TMB is asubstrate for horseradish peroxidase often used in ELISA. Reactionbetween the substrate and immobilized horseradish peroxidase (HRP)conjugated to antibodies in the ELISA wells produces a blue coloredsolution. After reaching the desired color intensity, the reaction isterminated by addition of the stop solution (acidic), which changes thesolution color from blue to yellow. Thus, the reactions were stoppedafter the incubation by adding 50 μL per well of kit-supplied StopSolution to each well and the absorbance read at 450 nm.

Cystatin C levels were measured with a commercially available kit (R&DSystems, Minneapolis, Minn.; catalogue number: DSCTC0). Briefly, urinesamples were diluted 1:3 using kit supplied calibrator diluent RD5-24.50 μL of each sample or kit standard control (recombinant human CysC)were added to wells of an assay plate in duplicate, each well containing100 μL of kit-supplied Assay Diluent RD1-52. After a two hour incubationat 4° C. in the refrigerator, wells were washed four times with 200 μLper well of wash solution. An enzymatically-labeled anti-CysC conjugatewas added at 200 μL per well and incubated for two hours at 4° C. in therefrigerator. Wells were washed four times with 200 μL per well of washsolution and developed by adding 200 μL per well of kit-supplied TMBSubstrate Solution (substrate for the enzyme of the anti-CysC conjugate)and incubated at room temperature in the dark for 30 minutes. Thereactions were stopped after the incubation by adding 50 μL per well ofkit-supplied Stop Solution to each well and the absorbance read at 450nm.

Clusterin levels were measured with a commercially available kit (R&DSystems, Minneapolis, Minn.; catalogue number: DCLU00). Briefly, urinesamples were diluted 1:3 using kit supplied calibrator diluent RD5T. 50μL of each sample or kit standard control (recombinant human clusterin)were added to wells of an assay plate in duplicate, each well containing100 μL of kit-supplied Assay Diluent RD1-19. After a two hour incubationat room temperature on the orbital shaker set at 500 rpm, wells werewashed four times with 200 μL per well of wash solution. Anenzymatically-labeled anti-clusterin conjugate was added at 200 μL perwell and incubated for two hours at room temperature on the orbitalshaker set at 500 rpm. Wells were washed four times with 200 μL per wellof wash solution and developed by adding 200 μL per well of TMBSubstrate Solution and incubated at room temperature in the dark for 30minutes. The reactions were stopped after the incubation by adding 50 μLper well of Stop Solution to each well and the absorbance read at 450nm.

TIMP-1 levels were measured with a commercially available kit (R&DSystems, Minneapolis, Minn.; catalogue number: DTM100). Briefly, urinesamples were diluted 1:2 using kit-supplied calibrator diluent RD5P. 50μL of each sample or kit standard control (recombinant human TIMP-1)were added to wells of an assay plate in duplicate, each well containing100 μL of kit-supplied Assay Diluent RD1X. After a two hour incubationat room temperature on the orbital shaker set at 500 rpm, wells werewashed three times with 200 μL per well of wash solution. Anenzymatically-labeled anti-TIMP-1 conjugate was added at 200 μL per welland incubated for two hours at room temperature on the orbital shakerset at 500 rpm. Wells were washed four times with 200 μL per well ofwash solution and developed by adding 200 μL per well of TMB SubstrateSolution and incubated at room temperature in the dark for 30 minutes.The reactions were stopped after the incubation by adding 50 μL per wellof Stop Solution to each well and the absorbance read at 450 nm.

β2M levels were measured with a commercially available kit (R&D Systems,Minneapolis, Minn.; catalogue number: DBM200). Briefly, urine sampleswere diluted 1:10 using kit supplied Sample Diluent. 20 μL of eachsample, kit controls or kit standards were added to wells in duplicate,containing 100 μL of a solution containing enzymatically-labeledanti-β2M conjugate. After a one hour incubation at room temperature,wells were washed six times with 200 μL per well of wash solution. Wellswere developed by adding 100 μL per well of TMB Substrate solution andincubated at room temperature in the dark for 15 minutes. The reactionswere stopped after the incubation by adding 100 μL per well of StopSolution to each well and the absorbance read at 450 nm.

Creatinine levels were measured with a commercially available kit (R&DSystems, Minneapolis, Minn.; catalogue number: KGE005). Briefly, urinesamples were diluted 1:20 using water and 50 μL of samples, kit controlsor kit standards were added to wells in duplicate, containing 100 μL ofthe kit-supplied Alkaline Picrate Solution. After a 30 minute incubationat room temperature, the absorbance at 490 nm was measured.

C5b-9 levels were measured with a commercially available kit (BDBiosciences, San Jose, Calif.; catalogue number: 558315) and an optEIAreagent set B (BD Biosciences, San Jose, Calif.; catalogue number:550534). Briefly, an anti-C5b-9 capture antibody was diluted 1:250 incoating buffer, 100 pL of which was added to each well of a 96 wellmaxisorp plate (Nunc; catalogue number: 439454) and incubated overnightat 4° C. in the refrigerator. Wells were washed three times with 200 μLper well of wash solution and blocked by adding 200 μL per well ofkit-supplied Assay Diluent for one hour at room temperature. Wells werewashed three times with 200 μL per well of wash solution and 100 μL ofurine samples or kit standards were added to wells in duplicate. After atwo hour incubation at room temperature, wells were washed three timeswith 200 μL per well of wash solution. 100 μL of the kit-supplied C5b-9Working Detector Antibody Solution was added to each well and incubatedfor one hour at room temperature. Wells were washed seven times with 200μL per well of wash solution and developed by adding 100 μL per well ofTMB Substrate Solution and incubated at room temperature in the dark for30 minutes. The reactions were stopped after the incubation by adding 50μL per well of Stop Solution to each well and the absorbance read at 450nm.

C5a levels were measured with a commercially available kit (BDBiosciences, San Jose, Calif.; catalogue number: 557965). Briefly, 100μL of urine samples or kit standards were added to wells in duplicatecontaining 50 μL of kit-supplied ELISA Diluent. After a two hourincubation at room temperature, wells were washed five times with 200 μLper well of wash solution. 100 μL of the kit-supplied C5a WorkingDetector Antibody Solution was added to each well and incubated for onehour at room temperature. Wells were washed seven times with 200 μL perwell of wash solution and developed by adding 100 μL per well of TMBSubstrate Solution and incubated at room temperature in the dark for 30minutes. The reactions were stopped after the incubation by adding 50 μLper well of Stop Solution to each well and the absorbance read at 450nm.

Plasma

Plasma samples were prepared as follows. Blood was collected in a 10 mLBD™ P100 tube (Becton Dickinson) containing EDTA. Whole blood wascentrifuged no later than 60 minutes after collection in a refrigeratedcentrifuge (set to maintain 4-8° C.) for 10 minutes at 3000 rpm. Plasmawas then obtained from the sample following centrifugation. Hemolyzedsamples were discarded.

The concentration of several analytes including Ba, prothrombin fragment1+2, thrombomodulin, vWF, sC5b-9, and C5a in plasma fractions of bloodcollected from the subjects was measured using commercially-availablekits as described briefly below.

Ba levels were measured with a commercially available kit (Quidel, SanDiego, Calif.; catalogue number: A033). Briefly, wells of an assay platewere washed three times with wash solution. Plasma samples were diluted1:1000 with kit-supplied specimen diluent and 100 μL of the dilutedplasma samples, kit controls and standards were added to wells induplicate. After a 60 minute incubation at room temperature, the wellswere washed five times with 200 μL per well of wash solution. 100 μL ofan enzymatically-labeled anti-Ba antibody conjugate were added to eachwell and incubated for sixty minutes at room temperature. After fivewashes with wash solution, 100 μL of TMB substrate was added to eachwell and incubated for fifteen minutes at room temperature protectedfrom light. The reaction was stopped with the addition of 100 μL perwell of stop solution and absorbance was read at 450 nm.

Prothrombin fragment 1+2 levels in EDTA plasma were measured with theEnzygnost F1+2 kit (Siemens Healthcare; catalogue number: OPBD03).Briefly, plasma samples were diluted 1:2 with sample buffer and 50 μL ofthe diluted samples, or standard (containing a known concentration ofrecombinant human prothrombin fragment 1+2) were added to wells. After a30 minute incubation at 37° C., the wells were washed three times with200 μL per well of wash solution. 100 μL of an enzymatically-labeledanti-prothrombin fragment 1+2 antibody conjugate were added to each welland incubated for 15 minutes at 37° C. After three additional washes,100 μL of chromagen substrate were added to each well and incubated 15minutes at room temperature protected from light. The reaction wasstopped by the addition of 100 μL of stop solution to each well andabsorbance read at 450 nm.

Levels of thrombomodulin (TM) in EDTA plasma were evaluated with the TMELISA kit (American Diagnostica, Stamford, Conn.; catalogue number:837). Briefly, plasma samples were diluted 1:4 with sample buffer and200 μL of diluted samples or standard (containing a known concentrationof recombinant TM) was added to wells. After a 60 minute incubation atroom temperature, wells of the assay plate were washed four times with200 μL/well of wash solution. A solution of an enzymatically-labeledanti-TM antibody was added (200 μL per well) and incubated for 30minutes at room temperature. After 4 washes, 200 μL of substrate wereadded to each well and the wells were incubated for 20 minutes at roomtemperature protected from light. The reaction was stopped with 100 μLof 0.5 M H₂SO₄ and the absorbance at 450 nm was measured.

Levels of von Willebrand Factor (vWF) activity were determined in EDTAplasma by an ELISA kit utilizing capture antibody specific for vWFcollagen binding sites (American Diagnostica; catalogue number: 885).Plasma samples and kit controls were diluted 1:20 with assay diluent and100 μL of the diluted samples and controls added to wells in duplicate.After a 60 minute incubation at room temperature, the wells were washed5 times with wash solution and 100 μL of an enzymatically-labeledanti-vWF conjugate were added to each well. The wells were incubated for15 minutes at room temperature and washed 5 times with wash solution.100 μL of TMB substrate (which, upon cleavage by the enzyme, generates adetectable signal) was added to each well. The wells were incubated for15 minutes at room temperature protected from light followed by theaddition of 100 μL of kit-supplied stop solution to each well.Absorbance was measured at 450 nm within 30 minutes of the addition ofstop solution.

Circulating levels of sC5b-9 were determined with a human C5b-9 ELISAset (BD Biosciences, San Diego, Calif.; catalogue number: 558315) and aBD optEIA reagent set B (BD Biosciences; catalogue number: 550534).Briefly, an anti-C5b-9 capture antibody was diluted 1:250 inkit-supplied coating buffer, 100 μL of which were added to wells of a 96well maxisorp plate (Nunc) and incubated overnight at 4° C. Followingthree washes in wash solution, wells were blocked with 200 μLkit-supplied assay diluent for one hour at room temperature. Following 3more washes with wash solution, 100 μL of the plasma samples (diluted1:100 in assay diluent) or standards (containing a known concentrationof purified sC5b-9) were added and incubated for two hours at roomtemperature. The wells were washed three times with wash solution and100 μL of working detector (which contains a biotin-labeled anti-C5b-9detection antibody and streptavidin-labeled horseradish peroxidasediluted 1:250 in assay diluent) added to each well. Following a one hourincubation at room temperature, the wells were washed seven times withwash solution and 100 μL of substrate TMB solutions added to each well.The reaction was allowed to develop for 30 minutes at room temperatureprotected from light. Following the addition of 50 μL of stop solutionto each well, absorbance was determined at 450 nm.

Circulating levels of C5a were determined with a sandwich ELISAutilizing the BD optEIA reagent set B (BD Biosciences; catalogue number:550534). All incubations were performed in the presence of futhan (BDBiosciences; catalogue number: 550236). Briefly, an anti-C5a captureantibody was diluted to 2 μg/mL in kit-supplied coating buffer, 100 μLof which were added to wells of a 96 well maxisorp plate (Nunc) followedby an overnight incubation at 4° C. Following three washes in washsolution, wells were blocked with 200 μL assay diluent for one hour atroom temperature. Following 3 more washes with wash solution, 50 μL ofplasma samples (diluted 1:5 in assay diluent) or standards (containing aknown concentration of C5a) were added and incubated for one hour atroom temperature. The wells were washed 4 times with wash solution and100 μL of working detector added to each well contains a biotin-labeledanti-C5a detection antibody and streptavidin-labeled horseradishperoxidase diluted 1:250 in assay diluent). Following an incubation forone hour at room temperature, wells were washed seven times with washsolution and 100 μL of substrate TMB solutions added to each well. Thereaction was allowed to develop for 30 minutes at room temperatureprotected from light. Following the addition of 50 μL of stop solutionto each well, absorbance was measured at 450 nm.

Serum

Serum samples were processed as follows. Blood was collected in a 10 mLvacutainer serum separating (SST) tube. The tube was inverted five timesand the blood allowed to clot at room temperature for at least 30minutes, but no more than two hours. The tube was subjected tocentrifugation at 1800 rcf with the brake on. Hemolyzed samples werediscarded.

The quantitative determination of various analytes in serum was carriedout using human Cytometric Bead Array (CBA) Flex Set Kits (CBA Flex Set;Becton Dickinson Biosciences, San Diego, Calif.), and acquired by flowcytometer (FACS LSR II, Becton Dickinson) according to themanufacturer's instructions. A Flex set capture bead is a single beadpopulation with distinct fluorescent intensity and is coated with acapture antibody specific for a soluble protein. Each bead population isgiven an alphanumeric position designation, indicating its positionrelative to other beads in the BD CBA Flex Set system. Beads withdifferent positions can be combined in assays to create a multiplexassay. In a Flex Set assay the capture bead, PE conjugated detectionreagent, and the standard or test samples are incubated together to formsandwich complexes.

Briefly, standards for each analyte were mixed and a serial dilution wasperformed using the assay diluent. Capture beads for each analyte wereprepared and pooled using Capture bead diluent for serum/plasma. Serumsamples were diluted appropriately and along with the standards wereincubated with the mixed capture beads in a total volume of 100 μL forone hour at room temperature. Detection phycoerythrin (PE) reagents weremixed for all analytes and were added to the tubes (50 μL). The sampleswere washed with wash buffer after an incubation of two hours at roomtemperature in the dark and were acquired by flow cytometer afterreconstitution of the pellet in the wash buffer.

The following bead sets were incubated with serum samples diluted 1:4 inkit-supplied assay diluent (wherein the biomarker protein specifiedindicates the capture antibody conjugated to the bead): IFN-γ (Bead E7;catalogue number: 558283); IL-12 p70 (Bead E5; catalogue number:558283); IL-1β (Bead B4; catalogue number: 558279); IL-6 (Bead A7;catalogue number: 558276); IL-8 (Bead A9; catalogue number: 558277);CXCL-9 (Bead E8; catalogue number: 558286); CXCL-10 (Bead B5; cataloguenumber: 558280); MCP-1 (Bead D8; catalogue number: 558287); VEGF (BeadB8; catalogue number: 558336); and sCD40L (Bead C7; catalogue number560305). The following bead sets were incubated with serum samplesdiluted 1:50 in kit-supplied diluent: ICAM-1 (Bead A4; catalogue number:560269); VCAM-1 (Bead D6; catalogue number: 560427); TNFR1 (Bead C4;catalogue number: 560156); E-selectin (Bead D9; catalogue number:560419); P-selectin (Bead D7; catalogue number: 560426); and CCL5 (BeadD4; catalogue number: 558324).

Example 2 Results

Markers of Ongoing Complement Activation

As summarized in Table 2 below, relative to the concentration in asample of biological fluid from healthy volunteers, the plasmaconcentration of complement component Ba and sC5b9 and the urineconcentration of C5a and sC5b-9 were elevated in the majority of aHUSpatients. See also FIG. 1.

TABLE 2 aHUS Biomarker n/N (%) elevated Protein at baseline P-valuePlasma Ba  35/35 (100.0) <0.0001 Plasma sC5b-9 37/38 (97.4) <0.0001Urine C5a 26/29 (89.7) 0.0007 Urine sC5b-9 23/27 (85.2) 0.0025 * “N”indicates the total number of patients evaluated for a given biomarker,and “n” indicates the number of those “N” patients with elevated levelsof the biomarker protein.

These results indicate that significant systemic alternative pathwaycomplement activation is ongoing in aHUS patients.

Following treatment with eculizumab, the mean levels (concentrations) ofthese aHUS biomarkers were reduced (FIGS. 1A-C). The mean levels ofurinary C5a and sC5b-9 are reduced significantly at between 1 to 2.5weeks following initiation of treatment and remained so. The meanpercentage reduction in urinary C5a levels was greater than 40% at week3 post-treatment and over 70% by week 6 (FIG. 1D). Urinary sC5b-9 levelswere reduced by over 60% by week 3 (FIG. 1E). These markers eventuallynormalized. Plasma Ba levels were also significantly reduced (p=0.0053)at around four to six weeks following treatment with eculizumab,suggesting that with time eculizumab reduces the initiation oramplification of the classical complement pathway (FIG. 1C). However,the mean percentage reduction in plasma Ba levels was around 10% at week6 and over 30% by week 12 (FIG. 1F).

The percentage of treated aHUS patients who experience normalizedcomplement biomarker protein levels over time is shown in FIGS. 2A-C.For example, as shown in FIG. 2B, 50% of treated aHUS patients exhibitnormalized levels of urinary sC5b-9 by 2.5 weeks post-treatmentinitiation with eculizumab. By 17 weeks post-initiation of treatment,79% of treated aHUS patients exhibited normalized sC5b-9 levels.However, Ba levels do not normalize in most patients (FIGS. 1C and 2C).These data indicate that even with eculizumab therapy there may be, insome patients, low level ongoing complement activation.

Markers of Platelet and Hemostatic Activation

As summarized in Table 3 below, relative to the concentration in asample of biological fluid from healthy volunteers, the serumconcentration of sCD40L and the plasma levels of prothrombin fragment1+2 and D-dimer were significantly elevated in the majority of aHUSpatients. See also FIGS. 3A-B.

TABLE 3 aHUS Biomarker n/N (%) elevated Protein at baseline P-valuesCD40L 36/38 (94.7) <0.0001 Prothrombin Fragment F1 + 2 36/38 (94.7)<0.0001 D-dimer 34/36 (94.4) =0.0002 * “N” indicates the total number ofpatients evaluated for a given biomarker, and “n” indicates the numberof those “N” patients with elevated levels of the biomarker protein.

The release of sCD40L is generally associated with platelet metabolismand activity. Prothrombin fragments F1+2 are generated during conversionof prothrombin to thrombin, whereas D-dimer is a fibrin degradationproduct indicating fibrinolysis.

Following treatment with eculizumab, the mean levels (concentrations) ofthese aHUS biomarkers were reduced. The mean levels of plasma levels ofF1+2 and D-dimer are reduced significantly at between 1 to 2.5 weeks(p=0.0078 and 0.0083, respectively) following initiation of treatmentand remained so. As shown in FIG. 3C, the mean percentage reduction inF1+2 was around 15% by week 3 and over 50% by week 12. The meanpercentage reduction in d-dimer levels was around 40% at week 6, butgreater than 60% by week 12. These data indicate that eculizumab therapyhas an immediate effect on the coagulation and fibrinolysis pathways. Asshown in FIGS. 4A-B, 32% of treated aHUS patients exhibit normalizationof F1+2 levels by week 26 post-initiation of treatment and 46% of thepatients have normalized levels of D-dimer. By contrast, sCD40L levelsremained elevated throughout the study.

Markers of Endothelial Cell Damage and/or Activation

As summarized in Table 4 below, relative to the concentration in sampleof biological fluid from healthy volunteers, the plasma concentration ofthrombomodulin and vWF and the serum concentration of VCAM-1 weresignificantly elevated in aHUS patients. See also FIGS. 5A-C.

TABLE 4 aHUS Biomarker n/N (%) elevated Protein at baseline P valueThrombomodulin 33/34 ( 97.1) <0.0001 VCAM-1 36/38 ( 94.7) <0.0001 VonWillebrand Factor 15/38 ( 39.5) <0.02 Antigen * “N” indicates the totalnumber of patients evaluated for a given biomarker, and “n” indicatesthe number of those “N” patients with elevated levels of the biomarkerprotein. n.s. indicates not significant.

High concentration of thrombomodulin and VCAM-1 in biological fluids ofaHUS patients indicates significant endothelial cell activation.Thrombomodulin is released in response to C3a, which further underscoresongoing complement activation in aHUS patients. vWF concentration isalso significantly elevated. vWF has a number of physiological rolesincluding platelet adhesion and coagulation and is also a marker ofendothelial damage and activation.

Following treatment with eculizumab, the mean levels (concentrations) ofthese aHUS biomarkers were reduced (FIGS. 5A-C). The mean levels ofthrombomodulin and VCAM-1 were reduced significantly from baseline byweek 17 (p=0.0007 and <0.0001, respectively) following initiation oftreatment (see FIGS. 6C and 6D). By week 26, levels of VCAM-1 and vWFhad also been reduced. However, while vWF normalized in ˜70% of treatedaHUS patients by week 17 post-initiation of treatment (FIG. 6B),thrombomodulin and VCAM-1 levels remained elevated. Interestingly, ofthe 10% of patients who normalized thrombomodulin levels (FIG. 6A), onlyone patient had both normalized thrombomodulin and vWF levels. Thesedata indicate that eculizumab therapy has a rapid and robust positiveeffect to correct endothelial cell damage and activation.

Markers of Inflammation

Table 5 (below) sets forth a series of analytes detected in plasmaand/or serum and indicates the percentage of aHUS patients in which therespective analytes were elevated prior to treatment with complementinhibitor therapy.

TABLE 5 aHUS Biomarker n/N (%) elevated Protein at baseline P valueSerum CXCL10 23/38 (60.5) P < 0.0001 Serum CXCL9 33/38 (86.8) P < 0.0001IL-18 19/38 (50.0) P < 0.0001 MCP-1 34/38 (89.5) P < 0.0001 TNFR1 38/38(100.0) P < 0.0001 VEGF 25/38 (65.8) P < 0.0001 IL-6 21/34 (61.8) P =0.0019 CCL5  4/38 (10.5) P = 0.0045 IFN-γ  5/34 (14.7) P = 0.0353 IL-822/38 (57.9) n.s. (P = 0.0640) ICAM-1  2/34 (5.9) n.s IL-1β  1/38 (2.6)n.s IL-12p70  2/34 (5.9) n.s * “N” indicates the total number ofpatients evaluated for a given biomarker, and “n” indicates the numberof those “N” patients with elevated levels of the biomarker protein. **The concentrations of the two analytes marked as “Serum” were measuredin serum. The concentrations of all other analytes in the Table weremeasured in plasma. n.s. indicates not significant.

Prior to therapy with eculizumab, patients with aHUS had elevated levelsof circulating inflammatory cytokines and chemokines including, e.g.,CXCL-10, CXCL-9, IL-18, TNFR1, MCP-1, VEGF, IL-6, and IL-8. Followinginitiation of treatment, however, TNFR1 was the earliest inflammatorymarker to be significantly reduced (by week 6, p=0.0012) (FIG. 7A). Meanconcentration of TNFR1 remained significantly lower than baseline at allsubsequent visits (P<0.0001), but only normalized in 6% of aHUS patients(FIG. 7B). Similarly, mean levels of CXCL10 were significantly reducedby week 26 (p=0.0055), but did not normalize in all aHUS patients (31%of patients did not normalize). By week 26, mean levels of IFN-γnormalized in approximately 50% of patients; however, mean levels ofserum IL-8 (p=0.01), CXCL-9 (p=0.01), IL-18 (p<0.0001) and VEGF(p<0.0001) remained elevated in most aHUS patients, as compared tonormal controls, and not significantly different from baseline. SerumIL-6 was significantly reduced (p=0.04) from baseline at week 26 andremained elevated at week 26 as compared to normal control.

By contrast, mean levels of CCL-5 were elevated significantly by week 17post-initiation of treatment and thereafter (p=0.0072 and 0.0021 atweeks 12-17 and week 26, respectively). In response to vascular injuryin mice, CCL5 is upregulated, which promotes selective T cellinfiltration as part of a vascular wound-healing response. See, e.g.,Rookmaaker et al. (2007) Am J Physiol Renal Physiol 293(2):F624-630.These data indicate that eculizumab therapy has a rapid and robustpositive effect on inflammation in many patients with aHUS, but that lowlevel inflammation may exist in these patients even during treatment.

Markers of Renal Tubular and Glomerular Injury

Table 6A (below) sets forth a series of analytes detected in urinecollected from patients and indicates the percentage of aHUS patients inwhich the respective analytes were elevated prior to treatment withcomplement inhibitor therapy.

TABLE 6A n/N (%) elevated Biomarker at baseline P value Beta-2Microglobulin (β2M) 20/28 ( 71.4) P < 0.0001 Clusterin 24/29 ( 82.8) P <0.0001 Cystatin C 18/29 ( 62.1) P = 0.0002 TIMP-1 22/29 ( 75.9) P =0.0003 FABP-1 22/29 ( 75.9) P = 0.0130 NGAL  5/29 ( 17.2) P = 0.0151 NAG 3/23 ( 13.0) P = 0.0413 CXCL10 2/29 ( 6.9) n.s. CXCL9  2/29 ( 6.9) n.s.KIM-1 2/29 ( 6.9) n.s. * “N” indicates the total number of patientsevaluated for a given biomarker, and “n” indicates the number of those“N” patients with elevated levels of the biomarker protein. n.s.indicates not significant.

Prior to treatment with eculizumab, low molecular weight molecules thatare normally filtered by the kidney were elevated in the urine ofpatients with aHUS including β2M, clusterin, cystatin C, and NAG.Molecules produced by renal tubular epithelial cells in response toinjury were also elevated, such as TIMP-1, NGAL and L-FABP. However,following treatment with eculizumab, CysC (p=0.0012) (FIG. 8A),clusterin (p=0.0446), and TIMP-1 (p=0.0353) are significantly reduced by1-2.5 weeks post-initiation of treatment and they remained significantlyreduced throughout the course of the study. NGAL (p=0.0003) (FIG. 8C),L-FABP (p=0.0366), and NAG (p=0.0369) were significantly reduced frombaseline by 4-6 weeks post-initiation of treatment and remained sothereafter. β2M was significantly reduced at 12-17 weeks (p=0.0008) andonwards (FIG. 8B). By week 26, mean urinary levels of all analytes hadnormalized in treated aHUS patients.

These data indicate that eculizumab therapy has a rapid, robust, anddurable positive effect redressing renal tubular and glomerular injuryexperienced by many patients with aHUS.

Summary

The following Table provides a summary of exemplary aHUS biomarkers(though not an exhaustive list), which are elevated in aHUS patientsprior to treatment with eculizumab, but are significantly reducedfollowing treatment with eculizumab. Also provided in the Table (Table6B) is the average time post-initiation of treatment with eculizumab inwhich significant reduction of the aHUS biomarker occurred.

TABLE 6B Biomarker Week 1-2.5 Week 4-6 Week 12-17 Week 26 U-C5a XU-C5b-9 X F1 + 2 X D-dimer X U-Cys-C X U-TIMP-1 X Plasma Ba X TNFR1 XU-CLU X U-NGAL X Thrombomodulin X VCAM X L-FABP X B2M X CXCL10 X IFN-γ XBaseline aHUS Marker Levels in aHUS Patients Receiving Dialysis and/orReceiving Kidney Transplant

Also assessed were the concentration of plasma and urine complement,inflammation, and renal injury markers in aHUS patients who receiveddialysis prior to therapy with eculizumab. As shown in Table 7 (below),the mean concentration of serum TNFR1, plasma Ba, C5b9, prothrombinfragments 1+2, β2M, clusterin, sC5b9, TIMP-1, NGAL, CysC, and C5a weresignificantly elevated in aHUS patients who underwent repeated dialysis(e.g., two or more times within 6 months prior to treatment) as comparedto aHUS patients that did not undergo repeated dialysis prior toenrollment in the study (prior to treatment). See also FIGS. 9A-E.

TABLE 7 Higher with Repeated Analyte Dialysis TNFR1 (serum)   p =<0.0001 β2m (urinary) p = 0.0009 Clusterin (urinary) p = 0.0020 Ba(plasma) p = 0.0021 C5b-9 (urinary) p = 0.0042 TIMP-1 (urinary) p =0.0070 NGAL (urinary) p = 0.0110 CysC (urinary) p = 0.020  F1 + 2(plasma) p = 0.0191 C5b-9 (plasma) p = 0.0476 C5a (urinary) p = 0.0477** The concentration of the one analyte marked “serum” was measured inserum. The concentration of analytes designated with “urinary” wasmeasured in urine, whereas the concentration of analytes labeled with“plasma” was measured in plasma obtained from the patients.

In addition, aHUS patients who had received a kidney transplant prior totreatment with eculizumab had lower urinary C5b-9 and urinary FABP-1 atbaseline as compared to patients who had not received a kidneytransplant.

Baseline aHUS Marker Levels Vis-à-Vis TMA Markers

Levels of some aHUS-associated biomarkers in some aHUS patientscorrelated with abnormal thrombotic microangiopathy (TMA) markers suchas reduced platelet counts, elevated LDH, and increased haptoglobinlevels. For example, aHUS patients with reduced platelet counts atbaseline (<150,000 per μL of blood), exhibited elevated levels ofurinary cystatin C (P=0.0276) and urinary clusterin (P=0.0401). SeeFIGS. 14A-B. aHUS patients having elevated LDH levels exhibitedincreased levels of VCAM-1 (P=0.0226) (FIG. 14C), d-dimer (P=0.0369)(FIG. 14D), IL-18, thrombomodulin, and TNFR1 (see below). Elevatedhaptoglobin levels were often present in aHUS patients having elevatedIL-18 levels.

Baseline aHUS Marker Levels in aHUS Patients Receiving Plasma Therapy

aHUS patients with repeated plasma therapy prior to treatment witheculizumab exhibited higher mean levels of urinary cystatin C atbaseline (see FIG. 15).

Correlations Between Biomarker Levels and Clinical Parameters

Platelets

An elevated level of CCL5 was positively correlated with higher plateletcounts at baseline (p=<0.0001; cc (correlation coefficient)=0.8106). Anelevated level of sCD40L was also correlated with higher platelet countsat baseline (p=<0.001; cc=0.6313).

Moreover, patients with normalized Ba levels following eculizumabtreatment show significantly higher platelet increases than patientswhose Ba levels remain elevated following treatment. See FIG. 13.

Estimated Glomerular Filtration Rate (eGFR), LDH, and Urinary Complement

A correlation was also observed between elevated plasma Ba levels andreduced eGFR (p<0.0001; cc=−0.7219). An elevated concentration of TNFR1in serum of aHUS patients prior to treatment was correlated with lactatedehydrogenase (LDH) levels (p=0.027; cc=0.3586), but more significantlycorrelated with lower eGFR (p<0.0001; cc=−0.6134). In addition, higherlevels of urinary complement components C5a and sC5b-9 and renal injurymarkers (02M, clusterin, cystatin C, NGAL, and TIMP-1) were moderatelycorrelated with lower eGFR (p=0.0002 to 0.0242; cc=−0.4286 to −0.6714).

Elevated levels of urinary sC5b-9, clusterin, and TIMP-1 were modestlycorrelated with proteinuria (p=0.0086 to 0.0284; cc=0.40 to 0.4788),whereas elevated levels of plasma Ba (p=0.0017; cc=0.517), β2M,clusterin, urinary sC5b-9, and cystatin C were correlated with increasedcreatinine in the urine of patients prior to treatment with eculizumab(p=0.0440-0.0018; cc=0.3982-0.6457).

First Clinical Presentation of aHUS

Also observed was a correlation between patients experiencing theirfirst aHUS manifestation and significantly elevated plasma D-dimerlevels or urinary FABP-1 at baseline (prior to eculizumab treatment)(see Table 8).

TABLE 8 Biomarker Elevated (n %) Single aHUS Multiple BiomarkerManifestation Manifestations p-value Plasma D-dimer 27 (100.0) 7 (77.8)0.0571 (μg/L) Urine FABP-1 (ng/mg normalized 19 (90.5)  3 (37.5) 0.0079to creatinine)

Smoldering Disease

Six of the aHUS patients involved in the study presented at enrollmentwith normalized hematologic parameters (including haptoglobin, LDH, andplatelet levels). However, these patients still showed evidence ofchronic inflammation and complement activation despite a stable clinicalpicture. The patients had significantly elevated levels of serum TNFR1(as shown in FIG. 10A) as well as significantly elevated levels ofthrombomodulin, Ba (FIG. 10B), prothrombin fragments 1+2 (FIG. 10E),VCAM-1 (FIG. 10C), and d-dimer (FIG. 10D). Similarly, patients withnormal (>150×10⁹ platelets/μL) platelet levels at baseline still showelevated levels of most biomarkers (e.g., Ba (FIG. 10F), VCAM-1 (FIG.10G), D-dimer (FIG. 10H), and F1+2 (FIG. 10I). Taken together, thesefindings indicate that, even for the subset of aHUS patients deemed tobe in clinical remission following treatment, there are likely ongoinglow levels of complement activity, coagulopathy, and inflammation.

Correlations Between Biomarker Levels and Clinical Outcomes

Hematologic Responses

Patients with complete hematologic responses show more dramaticreductions in TNFR1, urinary clusterin, and urinary complement levels(C5a and C5b-9) (FIG. 11). For example, 86% of patients exhibiting areduced concentration of these aHUS biomarker proteins attained acomplete hematologic response (normalization of platelets and LDH) byweeks 12-17 post-initiation of treatment with eculizumab. Moreover,these patients showed a greater mean percentage reduction in serumTNFR1, urinary clusterin, urinary C5a, and urinary C5b-9 than patientswho did not attain a complete hematologic response.

Also observed was that the rapidity of reduction in TNFR1 (e.g., by week12 as compared to week 17 or beyond) was correlated with completehematologic response (p=0.0008). The rate of normalization of D-dimerwas significantly associated with a complete hematologic response(p=0.0109; cc=6.26).

Furthermore, the data show that a significantly greater increase inplatelet counts at weeks 12-17 (p=0.0022) and week 26 (p=0.0110) wasachieved in eculizumab-treated aHUS patients having (at weeks 12-17 andweek 26, respectively) normalized plasma Ba concentrations. Improvementin platelets was also correlated with a significant reduction in meanF1+2 levels at week 4-6 (P=0.0148; cc=−0.4087) and week 12-17 (P=0.0073;cc=−0.4396) and more modestly with a reduction in d-dimer levels at week12-17 (P=0.0470; cc=−0.3381). Nevertheless, a subset of patients,despite demonstrating a greater increase in platelet counts at weeksfour through 26, continued to exhibit significantly elevated levels ofprothrombin fragments 1+2, thrombomodulin, urinary β2M, clusterin,TIMP-1, and cystatin C, suggesting ongoing underlying disease activity.

Analysis of the data collected from the study also revealed acorrelation between the change in other biomarker protein concentrationand platelet recovery. For example, the concentration of CCL5, MCP-1,and sCD40L were positively correlated with increased platelet counts ineculizumab-treated patients as shown in Table 9 below.

TABLE 9 Week following initiation of treatment with Correlationeculizumab Biomarker P-value coeff.   1-2.5 CCL-5 p < 0.0001 0.7419sCD40L P = 0.0141 0.3950 VEGF P = 0.0014 0.5002 4-6 CCL-5 p < 0.00010.7743 sCD40L p < 0.0001 0.6818 MCP-1 P = 0.0114 0.4169 12-17 CCL5 P =0.0003 0.5656 26 CCL-5 p < 0.0001 0.7845 sCD40L P = 0.0012 0.5398

Thrombomicroangiopathy (TMA)

Eculizumab-treated aHUS patients having a greater reduction in plasma Balevels more frequently achieved a complete TMA response (e.g.,normalization of hematologic parameters (e.g., platelet count and LDHlevels) and preservation of renal function). For example, 72.7% ofpatients attained a complete TMA response by weeks 12-17, and 85.29% ofthe patients achieved a complete TMA response by week 26. As shown inFIG. 12, these patients showed a greater mean percentage reduction inplasma Ba concentration than patients who did not attain a complete TMAresponse (p=0.0018 and p=0.006, respectively).

Post-Treatment eGFR

Also observed was a relationship between the reduction and/ornormalization of certain biomarkers and an improvement in eGFR. Forexample, a significantly greater improvement (Table 10) in eGFR (e.g.,eGFR≧15 mL/min/1.73 m² sustained for at least two consecutivemeasurements obtained at least four weeks apart) was observed amongpatients with normalized MCP-1, IL-6, and IFN-γ (at weeks 4-6);normalized VCAM-1, CXCL10, CXCL9, and Ba (at weeks 12-17), andnormalized Ba, urinary β2M, urinary CysC, vWF, D-dimer, clusterin,CXCL10, CXCL9, urinary FABP-1, and others (at week 26) (Table 10). Seealso FIG. 16.

TABLE 10 Week post- initiation of treatment w/ Normalized eculizumabBiomarker p value   1-2.5 * * 4-6 MCP-1 0.0002 IL-6 0.0251 VCAM-1 0.016612-17 VCAM-1 0.0003 CXCL-10 0.0071 Ba 0.0299 CXCL-9  0.0441 26 VCAM-1<0.0001 Cystatin C <0.0001 Ba 0.0002 U-β2m 0.0013 CXCL9  0.0027 CXCL100.0172 vWF 0.0052 D-dimer 0.0224 L-FABP 0.0230 Clusterin 0.0300 F1 + 20.0460

Example 3 Baseline Levels of Selected aHUS Biomarker Proteins in aHUSPatients

At baseline, prior to eculizumab treatment, substantial evidence ofsignificant complement activation, vascular inflammation/damage, andorgan injury was observed in aHUS patients regardless of use of plasmaexchange/plasma infusion or normal laboratory values for platelet count,Hp or LDH. As evidenced by the data set forth in Table 11, theconcentrations of aHUS biomarkers of complement activity, vascularinflammation, endothelial activation and damage, coagulation, and renalinjury were significantly elevated in aHUS patients compared to healthysubjects.

TABLE 11 Fold Median Level increase Bomarker at BL [range] n/N (%) overDisease (NHV range; (P Value vs. Elevated NHV Process units) NHV**) atBL at BL CAP Plasma Ba 2676.4 35/35 (100)  5.53 Activation (388.0-588.0 [935.0-3668.0] ng/mL) (<0.0001) Terminal U-05a 9.00 26/29 (89.7) 45Complement (0.0-0.7  [0.3-76.6] Activation ng/mg U-creat)   (0.0007)U-sC5b-9 30.50 23/27 (85.2) 305 (0.0-0.6  [0.2-665.7] ng/mg U-creat)  (0.0025) Inflammation sTNFR1 17616.85 38/38 (100)  18.71 (407.3-1391.3  [4008.5-54158.2] pg/mL) (<0.0001) Endothelial sVCAM-1659.75 36/38 (94.7) 1.99 Activation (159.2-444.7  [375.4-1865.5] ng/mL)(<0.0001) Endothelial TM 10 33/34 (97.1) 3.64 Damage (2.0-3.6 [3.4-24.1] ng/mL) (<0.0001) Coagulation F1 + 2 1017.55 36/38 (94.7)5.46  (82.9-305.5  [217.7-5774.0] pmol/L) (<0.0001) D-dimer 2735 34/36(94.4) 9.84 (157.0-395.9  [330.0-44100.0] μg/L)   (0.0002) Renal InjuryU-clusterm 1232.30 24/29 (82.8) 8.62  (5.7-437.1  [129.9-6091.2] ng/mgU-creat) (<0.0001) U-TIMP-1 23.8 22/29 (75.9) 39.67 (0.0-5.4 [1.4-230.4] ng/mg U-creat)   (0.0003) U-L-FABP-1 58.00 22/29 (75.9)48.33  (0.0-16.9   [3.7-1309.8] ng/mg U-creat)   (0.0130) U-β2m 18.420/28 (71.4) 46 (0.0-2.7  [0.4-127.7] μg/mg U-creat) (<0.0001)U-cystatin-C 1256.9 18/26 (69.2) 23.85  (0.3-301.3  [14.3-7189.6] ng/mgU-creat)   (0.0001) NHV means normal human value or concentration for agiven aHUS biomarker protein recited in the Table. “creat” meanscreatinine, the concentration of which is used to normalize certainbiomarker concentrations recited in the table. CAP refers to alternativepathway of complement (see above). “BL” refers to “baseline”, i.e.,prior to treatment with eculizumab. “N” is the total number of patientsanalyzed for a given disease process and biomarker. “n” is the number of“N” patients in which a given biomarker was elevated. “U” indicates thatthe analyte was measured in urine. * P values were calculated using aWilcoxon Rank Sum test, testing for a difference between groups.

In addition, the inventors observed that there was no statisticalsignificance between the baseline elevated levels of certain aHUSbiomarkers observed in patients who had received or were receivingplasma exchange (PE) or plasma infusion (PI) therapy as compared to thelevel of elevation of the aHUS biomarkers in patients who did notreceive PE or PI therapy. For example, the concentration of Ba, sTNFR1,sVCAM-1, and D-dimer were not reduced or normalized in patients who hadreceived PE/PI therapy (FIGS. 17A-D). Note that only 3 of 26 patientsanalyzed in the data presented in FIGS. 17A-D did not receive PE/PI. Themajority of patients (n=23) had elevated levels of Cystatin C, ascompared to normal healthy volunteers. Cystatin C being a renal injurymarker (glomerular injury), it is possible that the patients who did notreceive PE/PI had less damage to their kidneys and thus had reducedlevels of renal injury-related biomarker proteins in their urine.

Similarly, at baseline, prior to eculizumab therapy, the concentrationof protein markers of complement activation (e.g., Ba), inflammation(e.g., sTNFR1), endothelial cell activation (sVCAM-1), coagulation(D-dimer), and renal injury (cystatin-C) were elevated in patients withaHUS having normal platelet counts. See FIGS. 18A-E. And patients withnormal Hp and LDH levels showed evidence of ongoing complementactivation, inflammation, endothelial cell activation, coagulation andrenal injury (see FIGS. 19A-E).

In view of the foregoing, the concentration of biomarkers reflectingcomplement activity, vascular inflammation, endothelial activation anddamage, coagulation and renal injury were chronically elevated inpatients with aHUS compared to normal healthy subjects. Patients withaHUS receiving PE/PI showed strong evidence of significant ongoingcomplement activation, vascular inflammation, endothelial activation,coagulation and renal injury. While PE/PI may transiently maintainnormal platelet count and LDH in some patients, the above resultsdemonstrate that the underlying complement dysregulation and TMAprocesses persist. Despite normal laboratory values for platelet count,LDH, and Hp, these studies indicate that significant ongoing complementactivation, vascular inflammation, endothelial activation, coagulationand renal injury exist in aHUS patients.

Example 4 Effects of Sustained Treatment with Eculizumab on aHUSBiomarker Concentrations

The inventors observed that sustained eculizumab treatment inhibitschronic elevated complement activation and terminal complement mediatedrenal injury, and reduces inflammation, endothelial damage andthrombotic risk in patients with aHUS. For example, sustained eculizumabtreatment rapidly and completely inhibited terminal complementactivation as indicated by the reduction in the concentration of bothC5a and sC5b-9 (e.g., urinary C5a and sC5b-9). See FIGS. 20A-B. Atbaseline, patients with aHUS showed significant terminal complementactivation compared with NHV, despite use of PE/PI or normal plateletcounts in some patients. In fact, aHUS patients demonstrated 45-foldhigher urinary C5a and 305-fold higher urinary sC5b-9 levels than NHV.However, during sustained eculizumab treatment, all aHUS patientsdemonstrated rapid and potent terminal complement blockade, withcomplete normalization of pathogenic terminal complement activationproducts and no difference in levels relative to NHV.

Furthermore, sustained eculizumab treatment normalized the concentrationof biomarker proteins of renal injury (FIGS. 21A-C). Prior to initiatingeculizumab therapy, the majority of patients had elevated levels ofbiomarkers of: tubular interstitial injury and deterioration of renalfunction (e.g., L-FABP-1, ˜48 fold higher than NHV), glomerularfiltration (e.g., cystatin C, ˜24-fold higher than NHV), proximaltubular injury (e.g., clusterin, 8.6 fold higher than NHV). However,sustained treatment with eculizumab dramatically reduced the urinaryconcentrations of FABP-1 (by up to 100%), cystatin C (by up to 99%), andclusterin (by up to 98%). This reduction was significant across alltimepoints (P<0.0001 for all) and the reduced concentration of all renalinjury markers was no different than levels in NHV. Additional renalinjury markers (e.g., TIMP-1 and β2-microglobulin) also normalized (seeabove under Example 2). These results suggest that organ ischemia anddamage may be entirely terminal complement dependent and confirmsclinical data demonstrating that sustained inhibition ofcomplement-mediated TMA led to clinically meaningful eGFR improvementand discontinuation of dialysis.

Sustained treatment with eculizumab also significantly reducescomplement alternative pathway activation (see FIG. 22). All patientswith aHUS showed significant systemic CAP activation upstream of C5,with 5.5-fold higher levels of Ba compared with NHV, prior to eculizumabtreatment. However, following initiation of eculizumab therapy, theconcentration of upstream biomarkers of CAP activation (e.g., Ba levels)was reduced by 30% and reduction after week 4-6 was significant acrossall timepoints (p<0.005) as compared with the concentration of themarkers in NHV. Yet Ba levels did not normalize in aHUS patients treatedwith eculizumab, suggesting that CAP activation persists, reflecting theunderlying complement dysregulation in patients with aHUS. To be clear,though, terminal complement blockade with eculizumab protected patientsfrom the clinical consequences of ongoing CAP activation.

In addition, chronic treatment of aHUS patients with eculizumab resultedin significantly reduced concentrations of biomarkers associated withinflammation, endothelial activation, and tissue damage (FIGS. 23A-C).Serum sTNFR1 levels were elevated (18.7-fold higher than NHV levels) in100% of patients with aHUS at baseline. Sustained treatment witheculizumab significantly reduced sTNFR1 up to 94%. The reduction in theconcentration of these biomarkers at week 4-6 was significant across alltimepoints (P<0.0001). Soluble VCAM-1 and TM levels were elevatedin >95% of aHUS patients at baseline by 2-fold and 3.6-fold,respectively, as compared to NHV, demonstrating significant endothelialcell activation and damage prior to eculizumab therapy. TM and sVCAM-1concentrations were also significantly reduced during eculizumabtreatment. After week 12-17, reduction in the concentration ofbiomarkers of endothelial damage was significant across all latertimepoints (TM; P<0.0001), but still modestly elevated compared to NHV.Dramatically reduced soluble TM levels may reflect restoration ofmembrane bound TM, which is protective against thrombotic risk.

Finally, chronic treatment with eculizumab rapidly and significantlyreduced the concentration of biomarkers associated with thrombotic riskand coagulation (FIGS. 24A-B). The concentration of coagulationbiomarkers F1+2 and D-dimer were significantly elevated (5.5-fold and9.8 fold higher than NHV) at baseline in greater than 94% of patientswith aHUS (P<0.0001 and P=0.0002, respectively). Yet F1+2 and D-dimerwere significantly reduced at 2.5 weeks post initiation of treatmentwith eculizumab. The concentration of F1+2 decreased by up to 88%(P<0.05 for all timepoints) and the D-dimer concentration was reduced byup to 99% (P<0.0001 for all timepoints) with sustained eculizumabtreatment. However, these two markers remained modestly elevated overthe respective concentrations in normal healthy subjects.

CONCLUSIONS

In view of the foregoing data, the inventors were able to draw a numberof conclusions. First, at baseline, elevated levels of all thromboticmicroangiopathy (TMA) biomarkers were evident in patients with aHUS ascompared to the levels in samples from normal healthy volunteers (NHV).In all patient groups—including those receiving PE/PI or those withnormal platelets, Hp or LDH—patients with aHUS demonstrated significantelevation, over NHVs, in measures of: terminal complement activation(45-305 fold higher than NHV levels); vital organ damage; alternativepathway of complement activation (e.g., as represented by Ba levels;5.5-fold higher than NHV levels); vascular inflammation; endothelialactivation and damage; and coagulation.

Sustained eculizumab treatment of aHUS patients significantly reducedand normalized highly elevated markers of terminal complementactivation. Inhibition of terminal complement activation with eculizumabalso dramatically reduced and normalized markers of organ damage.Upstream biomarkers of alternative pathway activation were alsosignificantly reduced, but did not normalize. And low levels ofalternative pathway activation persisted in treated patients, reflectingthe underlying complement dysregulation in patients with aHUS. Thatsaid, the data clearly indicate that terminal complement blockade witheculizumab protects aHUS patients from the clinical consequences ofongoing alternative pathway activation.

Moreover, sustained eculizumab treatment also resulted in: (i)significant and sustained reduction of markers of vascular inflammation(by up to 94%); (ii) significant inhibition of markers of endothelialactivation (by up to 60%); (iii) significant and sustained reduction inmarkers of endothelial damage (by up to 77%) to near normal levels,demonstrating a clear relationship between terminal complementactivation and endothelial damage; and (iv) marked reduction (by up to99%) of the concentration of biomarkers of thrombotic risk, likelydecreasing the potential for clot formation and thus reducing incidenceof TMA in these patients. The inventors conclude, while not being boundby any theory or mechanism of action, that inhibition of terminalcomplement activation with eculizumab must be sustained, as loss ofterminal complement inhibition in aHUS would lead to a rapid increase inseverely amplified terminal complement activation, subsequently leadingto: increase in underlying subclinical endothelial activation,significant acceleration of endothelial damage, marked increase inthrombotic risk, and an early and ongoing risk of catastrophic vascularischemia and vital organ damage. Moreover, these data indicate that therenal injury, vascular inflammation, and endothelial damage andactivation are in whole or in part dependent on terminal complementactivity, which activity is effectively and safely inhibited usingeculizumab.

While the present disclosure has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of thedisclosure. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, orprocess step or steps, to the objective, spirit and scope of the presentdisclosure. All such modifications are intended to be within the scopeof the disclosure.

What is claimed is:
 1. A method for treating a patient having atypicalhemolytic uremic syndrome (aHUS) whose blood or urine has beendetermined to contain elevated levels at least two aHUS-associatedbiomarker proteins selected from the group consisting of TNFR1, MCP-1,IFN-γ, IL-6, a proteolytic fragment of complement component factor B,soluble C5b9 (sC5b9), prothrombin fragment F1+2, d-dimer,thrombomodulin, VCAM-1, von Willebrand Factor (vWF), complementcomponent C5a, β2 microglobulin (β2M), clusterin, cystatin C, NAG,TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), albumin, CXCL9,KIM-1 and CCL5, soluble CD40 ligand (sCD40L), ICAM-1, IL-1 beta, IL-12p70, IL-8, and vascular endothelial cell growth factor (VEGF), themethod comprising administering to the patient an inhibitor ofcomplement C5 in an amount and with a frequency sufficient to reduce theconcentration of the at least two aHUS-associated biomarker proteinscompared to the concentration measured in the patient's blood or urineprior to treatment with the complement C5 inhibitor.
 2. The method ofclaim 1, wherein the subject: (a) has received dialysis at least oncewithin the three months immediately prior to treatment with thecomplement C5 inhibitor; or (b) is experiencing a first acute aHUSmanifestation.
 3. The method of claim 1, wherein the reducedconcentration of the at least two aHUS-associated biomarker proteinsoccurs within two weeks or two months after the first administration ofthe complement C5 inhibitor.
 4. The method of claim 1, wherein thesubject is chronically treated with a complement C5 inhibitor.
 5. Themethod of 1, wherein the complement C5 inhibitor is selected from thegroup consisting of a small molecule, a polypeptide, a polypeptideanalog, a peptidomimetic, and an aptamer.
 6. The method of 1, whereinthe complement C5 inhibitor is selected from the group consisting ofMB12/22, MB12/22-RGD, ARC187, ARC1905, SSL7, and OmCI.
 7. The method ofclaim 1, wherein the complement C5 inhibitor is an antibody, or anantigen-binding fragment thereof.
 8. The method of claim 7, wherein theantibody, or antigen-binding fragment thereof, is selected from thegroup consisting of a humanized antibody, a recombinant antibody, adiabody, a chimerized or chimeric antibody, a monoclonal antibody, adeimmunized antibody, a fully human antibody, a single chain antibody,an Fv fragment, an Fd fragment, an Fab fragment, an Fab′ fragment, andan F(ab′)₂ fragment.
 9. The method of claim 7, wherein the antibody, orantigen-binding fragment thereof, binds to complement component C5 andinhibits cleavage of C5 into fragments C5a and C5b.
 10. The method ofclaim 9, wherein the antibody is eculizumab or a variant of eculizumab.11. The method of claim 10, wherein the antigen-binding fragment ispexelizumab.
 12. The method of claim 1, wherein at least one of theaHUS-associated biomarkers is selected from the group consisting of:proteolytic fragment Ba of factor B, TNFR1, VCAM-1, D-dimer,thrombomodulin, and cystatin C.
 13. The method of claim 1, wherein theat least two aHUS-associated biomarker proteins are measured using animmunoassay, such as an enzyme-linked immunosorbent assay (ELISA) or aradioimmunoas say (RIA).
 14. The method of claim 1, wherein thebiological fluid is blood, a blood fraction, or urine.
 15. The method ofclaim 14, wherein the blood fraction is serum or plasma.
 16. The methodof claim 1, wherein: (i) the concentration of at least oneaHUS-associated biomarker is measured in two or more types of biologicalfluid; or (ii) the concentration of a first of the at least twoaHUS-associated biomarker proteins is measured in one type of biologicalfluid and the concentration of a second of the at least two aHUSbiomarker proteins is measured in a second type of fluid.
 17. The methodof claim 1, wherein: (i) the concentration of CXCL9, IFN-γ, MCP-1, CCL5,sCD40L, or sTNFR1 in the serum of the subject is determined; (ii) theconcentration of at least one of (32 microglobulin (β2M), clusterin,cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1),CXCL9, albumin, and KIM-1 in the urine of the subject is determined;(iii) the concentration of NGAL, a proteolytic fragment of complementcomponent factor B, soluble C5b9 (sC5b9), prothrombin fragment F1+2,D-dimer, thrombomodulin, or von Willebrand Factor (vWF) in the plasma ofthe subject is determined; and/or (iv) the concentration of Ba in plasmaobtained from the subject is determined.
 18. The method of claim 1,wherein the normal concentration of sTNFR1 is less than 2000 pg/mL. 19.The method of claim 1, wherein the concentration of sTNFR1 in thebiological sample is deemed elevated when it is: (i) at least two foldgreater than the normal concentration of sTNFR1; or (ii) at least 10,000μg/mL.